Skip to main content
. 2015 Aug 10;6:7904. doi: 10.1038/ncomms8904

Figure 4. PLK1 phosphorylates the Ser1359 residue of RSF1.

Figure 4

(a) RSF1 KO HeLa cells were transfected with indicated RSF1-C1 constructs and treated with paclitaxel for 16 h. Chromatin fractions of mitotic cell lysates were analysed by immunoblotting. (b,c) Recombinant His-PLK1 and GST-RSF1 (FL, C2 and N3) proteins were incubated at 30 °C for 30 min in the presence of γ32P-ATP. Incorporation of 32P into RSF1 protein was visualized by autoradiography. The asterisk indicates the hyper-phosphorylated form of RSF1-C2. (d) The RSF1-C1S1359A or RSF1-C1S1359D plasmids were transfected into RSF1 KO cells, and mitotic lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting. (e) RSF1 KO cells were introduced with indicated RSF1 constructs and treated with nocodazole for 4 h. Floating mitotic cells were subjected to chromosome spread immunostaining. Scale bar, 5 μm. (f) RSF1 KO HeLa cells were transfected with indicated RSF1-C1-V5 constructs and treated with paclitaxel for 16 h. RSF1-C1 protein immunopurified with anti-V5 antibody was incubated with recombinant GST-PLK1 protein in the in vitro kinase assay. See full blots Supplementary Fig. 8.