Figure 2.
Figure 2. Phosphorylation of ADD1 at Ser12 and Ser355 by CDK1 is crucial for ADD1 association with mitotic spindles. (A) HeLa cells transiently expressing FLAG-ADD1 and mutants were stained for FLAG-ADD1, α-tubulin, and DNA. (left) Representative confocal images from the cells in metaphase are shown, which were from a single experiment out of three repeats (n > 160). (right) Graphs show the relative fluorescence intensity (F.I.) of the lines that were scanned by confocal microscopy. a.u., arbitrary unit. Bar, 5 µm. (B) The percentage of FLAG-ADD1 association with mitotic spindles in the total number of mitotic cells counted was measured (n > 160). Values (means ± SD) are from three independent experiments. *, P < 0.05; **, P < 0.01. (C) FLAG-ADD1 and mutants were transiently expressed in HEK293 cells. The cells were synchronized in the G2/M phase or remained asynchronized (Async) before they were lysed. FLAG-ADD1 and mutants were immunoprecipitated (IP) by anti-FLAG, and the immunocomplexes were analyzed by immunoblotting (IB) with antibodies to ADD1 pS12 or pS355. S355-phosphorylated ADD1 is indicated by a white asterisk. WCL, whole-cell lysates. (D) HeLa cells were synchronized in the G2/M phase or remained asynchronized before they were lysed. Endogenous ADD1 was immunoprecipitated by anti-ADD1, and the immunocomplexes were analyzed by immunoblotting with antibodies to ADD1 pS12 or pS355. (E) HeLa cells were synchronized in the G2/M phase (+) or remained asynchronized (−). The cells were treated with 10 µM of the CDK1 inhibitor RO-3306 for 1 h before they were lysed. Endogenous ADD1 was immunoprecipitated by anti-ADD1, and the immunocomplexes were analyzed by immunoblotting with antibodies to ADD1 pS12 or pS355. S355-phosphorylated ADD1 is indicated by a white asterisk. (F and G) Purified T7-tagged ADD1 head domain (T7-ADD1 head) or the mutant with a deletion of the tail domain (Δtail) were incubated with recombinant CDK1 in the presence (+) or absence (−) of ATP for 30 min. The phosphorylation of ADD1 at S12 and S355 was analyzed by immunoblotting with antibodies to ADD1 pS12 or pS355.

Phosphorylation of ADD1 at Ser12 and Ser355 by CDK1 is crucial for ADD1 association with mitotic spindles. (A) HeLa cells transiently expressing FLAG-ADD1 and mutants were stained for FLAG-ADD1, α-tubulin, and DNA. (left) Representative confocal images from the cells in metaphase are shown, which were from a single experiment out of three repeats (n > 160). (right) Graphs show the relative fluorescence intensity (F.I.) of the lines that were scanned by confocal microscopy. a.u., arbitrary unit. Bar, 5 µm. (B) The percentage of FLAG-ADD1 association with mitotic spindles in the total number of mitotic cells counted was measured (n > 160). Values (means ± SD) are from three independent experiments. *, P < 0.05; **, P < 0.01. (C) FLAG-ADD1 and mutants were transiently expressed in HEK293 cells. The cells were synchronized in the G2/M phase or remained asynchronized (Async) before they were lysed. FLAG-ADD1 and mutants were immunoprecipitated (IP) by anti-FLAG, and the immunocomplexes were analyzed by immunoblotting (IB) with antibodies to ADD1 pS12 or pS355. S355-phosphorylated ADD1 is indicated by a white asterisk. WCL, whole-cell lysates. (D) HeLa cells were synchronized in the G2/M phase or remained asynchronized before they were lysed. Endogenous ADD1 was immunoprecipitated by anti-ADD1, and the immunocomplexes were analyzed by immunoblotting with antibodies to ADD1 pS12 or pS355. (E) HeLa cells were synchronized in the G2/M phase (+) or remained asynchronized (−). The cells were treated with 10 µM of the CDK1 inhibitor RO-3306 for 1 h before they were lysed. Endogenous ADD1 was immunoprecipitated by anti-ADD1, and the immunocomplexes were analyzed by immunoblotting with antibodies to ADD1 pS12 or pS355. S355-phosphorylated ADD1 is indicated by a white asterisk. (F and G) Purified T7-tagged ADD1 head domain (T7-ADD1 head) or the mutant with a deletion of the tail domain (Δtail) were incubated with recombinant CDK1 in the presence (+) or absence (−) of ATP for 30 min. The phosphorylation of ADD1 at S12 and S355 was analyzed by immunoblotting with antibodies to ADD1 pS12 or pS355.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal