Ox domain were generated by PCR amplification of Plk1 from mCherrytagged constructs and cloned into pQCXIN-Flag using standard restriction digest and ligation procedures. Plk1 fusion constructs were made using a modified USER cloning strategy. First, the Plk1 kinase domain was PCR TL32711 cost amplified and inserted into pQCFIN using standard restriction digest and ligation procedures. Next, the XbaI restriction site was removed by digest with XbaI, overhang removal by T4 DNA polymerase and blunt-end ligation. A 55-base double-stranded USER acceptance sequence containing XbaI and Nt.BbvCI restriction sites was inserted c-terminal to Plk1. The plasmid was then linearized by XbaI and NtBbvCI digest, creating 8-base overhangs. Kif2c, H2B and Dsn1 were PCR amplified with primers containing 8-base complementary sequences with a terminal uracil. Products were digested with USER enzyme and ligated into pQCFINPlk1C-USER vectors according to manufacturer’s protocol. Kif2c localization mutants were made by PCR amplification of amino acids 140-726 or site-directed mutagenesis using a QuikChange protocol. Constructs were fully sequenced to verify integrity. See Supplementary Author Manuscript Author Manuscript Author Manuscript Author Manuscript Nat Chem Biol. Author manuscript; available in PMC 2016 October 04. Lera et al. Page 11 Immunoblotting, Immunoprecipitation and Kinase Assays Author Manuscript Author Manuscript Author Manuscript Author Manuscript For all experiments, cells were challenged with 0.2 g/ml nocodazole for 19 h and mitotic cells were collected by shakeoff, pelleted, frozen down and stored at -80C prior to use. BubR1 detection included both mitotic and non-mitotic cells after 19 h nocadazole treatment. Immunoblotting–Cell pellets were lysed in buffer containing phosphatase inhibitors, 1mM PMSF, 1x protease inhibitor cocktail and 1 mM dithiothreitol. Proteins were separated by SDS-PAGE, transferred to Immobilon PVDF membrane, and blocked for 30 min in 4% milk and 0.1% Tween-20 Tris buffered saline ph 7.4. Membranes were incubated with gentle agitation for 2 h at room temperature with primary antibodies diluted 
in TBST+milk, washed 3x with TBST, incubated for 1 h at room temperature in secondary antibodies conjugated to horse radish peroxidase diluted 1:10,000 in TBST+milk. Membranes were washed and developed with luminol/peroxide and R-7128 web visualized with film. All results were obtained from single gels. To simultaneously probe for the protein of interest and the loading marker, the membrane was divided in two after transfer and incubated in separate antibody solutions. When identical-sized proteins prevented membrane division, the membrane was first probed for the protein of interest, stripped in an acidic glycine wash, rinsed in deionized H2O, and then reprobed for the loading marker. Immunoprecipitation and in vitro kinase assays–Cell lysates were prepared as above with up to 1 mg total protein used for pulldown. Protein inputs were adjusted to promote equivalent quantities after pulldown. Flag-construct lysates were immunoprecipitated with anti-Flag 
affinity gel for 1 h at 4C. mCherry-construct lysates were incubated for 3 h with 5 g/mL dsRed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19856273 antibody, followed by immunoprecipitation with a 1:1 mixture protein A and protein G sepharose beads for 1 h at 4C with gentle rotation. The beads/gel were washed three times with lysis buffer and then once with kinase buffer. Fifteen percent of immunoprecipitated complexes were removed for detection of b.Ox domain were generated by PCR amplification of Plk1 from mCherrytagged constructs and cloned into pQCXIN-Flag using standard restriction digest and ligation procedures. Plk1 fusion constructs were made using a modified USER cloning strategy. First, the Plk1 kinase domain was PCR amplified and inserted into pQCFIN using standard restriction digest and ligation procedures. Next, the XbaI restriction site was removed by digest with XbaI, overhang removal by T4 DNA polymerase and blunt-end ligation. A 55-base double-stranded USER acceptance sequence containing XbaI and Nt.BbvCI restriction sites was inserted c-terminal to Plk1. The plasmid was then linearized by XbaI and NtBbvCI digest, creating 8-base overhangs. Kif2c, H2B and Dsn1 were PCR amplified with primers containing 8-base complementary sequences with a terminal uracil. Products were digested with USER enzyme and ligated into pQCFINPlk1C-USER vectors according to manufacturer’s protocol. Kif2c localization mutants were made by PCR amplification of amino acids 140-726 or site-directed mutagenesis using a QuikChange protocol. Constructs were fully sequenced to verify integrity. See Supplementary Author Manuscript Author Manuscript Author Manuscript Author Manuscript Nat Chem Biol. Author manuscript; available in PMC 2016 October 04. Lera et al. Page 11 Immunoblotting, Immunoprecipitation and Kinase Assays Author Manuscript Author Manuscript Author Manuscript Author Manuscript For all experiments, cells were challenged with 0.2 g/ml nocodazole for 19 h and mitotic cells were collected by shakeoff, pelleted, frozen down and stored at -80C prior to use. BubR1 detection included both mitotic and non-mitotic cells after 19 h nocadazole treatment. Immunoblotting–Cell pellets were lysed in buffer containing phosphatase inhibitors, 1mM PMSF, 1x protease inhibitor cocktail and 1 mM dithiothreitol. Proteins were separated by SDS-PAGE, transferred to Immobilon PVDF membrane, and blocked for 30 min in 4% milk and 0.1% Tween-20 Tris buffered saline ph 7.4. Membranes were incubated with gentle agitation for 2 h at room temperature with primary antibodies diluted in TBST+milk, washed 3x with TBST, incubated for 1 h at room temperature in secondary antibodies conjugated to horse radish peroxidase diluted 1:10,000 in TBST+milk. Membranes were washed and developed with luminol/peroxide and visualized with film. All results were obtained from single gels. To simultaneously probe for the protein of interest and the loading marker, the membrane was divided in two after transfer and incubated in separate antibody solutions. When identical-sized proteins prevented membrane division, the membrane was first probed for the protein of interest, stripped in an acidic glycine wash, rinsed in deionized H2O, and then reprobed for the loading marker. Immunoprecipitation and in vitro kinase assays–Cell lysates were prepared as above with up to 1 mg total protein used for pulldown. Protein inputs were adjusted to promote equivalent quantities after pulldown. Flag-construct lysates were immunoprecipitated with anti-Flag affinity gel for 1 h at 4C. mCherry-construct lysates were incubated for 3 h with 5 g/mL dsRed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19856273 antibody, followed by immunoprecipitation with a 1:1 mixture protein A and protein G sepharose beads for 1 h at 4C with gentle rotation. The beads/gel were washed three times with lysis buffer and then once with kinase buffer. Fifteen percent of immunoprecipitated complexes were removed for detection of b.
