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. 1998 Aug 4;95(16):9301–9306. doi: 10.1073/pnas.95.16.9301

Figure 3.

Figure 3

Mutations in the polo-box do not impair Plk kinase activity in vitro. The cdc5–1 (KKY921–2B) cells bearing various YCplac111-GAL1-HA-PLK constructs were cultured under inducing conditions for 10 hr and were harvested. The lysates were centrifuged at 15,000 × g for 30 min to clarify heavy cellular materials. From 500 μg of cellular proteins present in the S15 fraction, the wild-type and mutant forms of HA-Plk were immunoprecipitated and subjected to in vitro kinase assays. (Upper) HA-tagged Plk was immunoprecipitated with affinity-purified anti-Plk antibody, was electrophoresed, and was detected on membranes by Western analysis with anti-HA antibody. (Lower) HA-tagged Plk was immunoprecipitated with anti-Plk antibody, and in vitro kinase assays were performed as described by using casein as a substrate (19). Vector, YCplac111-GAL1; K82M, YCplac111-GAL1-HA-PlkK82M; WT, YCplac111-GAL1-HA-Plk; W414F, YCplac111-GAL1-HA-PlkW414F.