Figure 5. USP44 is required for timely centrosome separation.

(A and B) MEFs transduced with the indicated USP44 constructs were subjected to immunoprecipitation using the indicated antibodies. Precipitates were immunoblotted using antibodies against the indicated proteins. Asterisk indicates IgG light chain. (C) N-terminally 6xHis-tagged USP44, GST, or GST–centrin 2 was expressed in bacteria, and resulting extracts were mixed, followed by affinity purification with glutathione agarose. Precipitates were immunublotted using the indicated antibodies. (D and E) Confocal immunofluorescence microscopy was performed on methanol-fixed MEFs transduced with USP44^Cherry using antibodies against the indicated proteins. Scale bar: 5 μm; insets, 1 μm. (F) The level of centrin 2/3 was determined in asynchronous MEFs of the indicated genotypes. Extracts were immunoblotted using antibodies for the indicated proteins. (G) Example of an Usp44-null cell with a supernumerary centrin signal at one spindle pole. Scale bar: 5 μm. (H) Incidence of one or more extra centrin signals in at least one spindle pole in MEFs of the indicated genotypes. Graph represents the mean ± SEM from 3 lines per genotype, 20 cells per line. *P < 0.05, unpaired t test.