Figure 8.
Figure 8. Cks enhances destruction of the N-terminal cyclin B1 fragment. (A) Cells expressing cyclin B1–Venus (lane 2), the first 86 amino acids of cyclin B1 fused to Venus (cycB1-N-Venus; lane 3), or the same N-terminal cyclin B1 fragment fused to Cks1 and Venus (cycB1-N-Cks1-Venus; lane 4) was synchronized in the spindle checkpoint and mitotic cells collected by mitotic shake off. GFP IPs on equal amounts of protein precipitated the Venus fusion proteins, which were blotted to examine coprecipitation of the indicated proteins (bottom). (B) Spindle checkpoint–arrested mitotic cells expressing cyclin B1–N–Venus or cyclin B1–N–Cks–Venus were collected, and lysates were subjected to sucrose gradient centrifugation. Fractions were blotted and probed with antibodies against the indicated proteins. (C) U2OS cells were cotransfected with vectors encoding cyclin B1–N–cerulean (cycB1-N) and cyclin B1–N–Cks–Venus (cycB1-N-Cks1) and followed as they progressed through mitosis. DIC, differential interference contrast. Bar, 10 µM. (D) Venus- and cerulean-tagged versions of cyclin B1–N or cyclin B1–N–Cks1 were cotransfected in U2OS cells. Cells either expressing cyclin B1–N or cyclin B1–N–Cks1 were followed during mitosis, and fluorescence levels were measured at NEB and plotted over time. For comparison, cells were also simultaneously transfected with three differently colored constructs, cyclin B1–N–Venus, cyclin B1–N–Cks1–cerulean, and a full-length red fluorescent cyclin B1 construct, cyclin B1–Cherry. Fluorescence levels of two triple-colored cells passing mitosis and performing a normal anaphase were included in the graph as a reference. Results of two independent experiments are shown. M, metaphase onset; A, anaphase onset. Comparable differences in destruction of the cyclin B1 reporters were found in >30 cells in four independent experiments. We found no significant effect of the different fluorescent tags on the destruction rates of the cyclin B1 fragments (Fig. S2 C). Markers are given in kilodaltons. WCE, whole cell extract.

Cks enhances destruction of the N-terminal cyclin B1 fragment. (A) Cells expressing cyclin B1–Venus (lane 2), the first 86 amino acids of cyclin B1 fused to Venus (cycB1-N-Venus; lane 3), or the same N-terminal cyclin B1 fragment fused to Cks1 and Venus (cycB1-N-Cks1-Venus; lane 4) was synchronized in the spindle checkpoint and mitotic cells collected by mitotic shake off. GFP IPs on equal amounts of protein precipitated the Venus fusion proteins, which were blotted to examine coprecipitation of the indicated proteins (bottom). (B) Spindle checkpoint–arrested mitotic cells expressing cyclin B1–N–Venus or cyclin B1–N–Cks–Venus were collected, and lysates were subjected to sucrose gradient centrifugation. Fractions were blotted and probed with antibodies against the indicated proteins. (C) U2OS cells were cotransfected with vectors encoding cyclin B1–N–cerulean (cycB1-N) and cyclin B1–N–Cks–Venus (cycB1-N-Cks1) and followed as they progressed through mitosis. DIC, differential interference contrast. Bar, 10 µM. (D) Venus- and cerulean-tagged versions of cyclin B1–N or cyclin B1–N–Cks1 were cotransfected in U2OS cells. Cells either expressing cyclin B1–N or cyclin B1–N–Cks1 were followed during mitosis, and fluorescence levels were measured at NEB and plotted over time. For comparison, cells were also simultaneously transfected with three differently colored constructs, cyclin B1–N–Venus, cyclin B1–N–Cks1–cerulean, and a full-length red fluorescent cyclin B1 construct, cyclin B1–Cherry. Fluorescence levels of two triple-colored cells passing mitosis and performing a normal anaphase were included in the graph as a reference. Results of two independent experiments are shown. M, metaphase onset; A, anaphase onset. Comparable differences in destruction of the cyclin B1 reporters were found in >30 cells in four independent experiments. We found no significant effect of the different fluorescent tags on the destruction rates of the cyclin B1 fragments (Fig. S2 C). Markers are given in kilodaltons. WCE, whole cell extract.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal