Figure 5.
Figure 5. Analysis of H2B S6 phosphorylation networks. (A) HeLa cells were transfected with a Dharmacon siRNA library against kinase and kinase-related genes. After 42 h, cells were treated with 200 nM nocodazole for 6 h and prepared for immunofluorescence staining against phosphorylated H2B S6. Phosphorylation intensities were quantified using a wide field Nikon microscope (Eclipse Ti-E) and high-content analysis software. Volcano plot shows standard deviations of average scores from mean H2B S6 phosphorylation intensity (x axis) and their statistical significance (P values; y axis). Statistical analysis was performed using GraphPad Prism. Aurora B, BubR1, and CDK1 are shown in red. (B) GFP-BubR1 and cyclin B1-Venus were expressed in 293T cells in the absence or presence of Myc-INCENP and HA-Aurora B WT or kinase dead (KD). Cells were lysed with SDS sample buffer and analyzed by SDS-PAGE and Western blotting using the indicated antibodies. (C) HeLa cells were arrested at prometaphase with nocodazole for 16 h and released in the presence of MG132 together with various concentrations of the indicated kinase inhibitors. Cells were lysed with SDS sample buffer and further analyzed by immunoblotting as shown. (D) Different plasmids encoding the indicated proteins, including constitutively active (AF) or kinase dead (KD) kinases, were expressed in 293T cells as shown. Aurora B was immunoprecipitated with HA antibodies and CDK1 with anti-Flag M2 affinity gel for a subsequent in vitro kinase assay with recombinant histone octamers as substrate. Samples were analyzed by SDS-PAGE, CBB staining, and immunoblotting using the indicated antibodies; unspecific IgG bands are indicated by asterisks. (E) Cyclin B1-Venus and Flag-tagged CDK1 forms were coexpressed in 293T cells. Cells were lysed with NP-40 buffer, and CDK1 was immunoprecipitated with anti-Flag M2 affinity gel for in vitro kinase assay using H2B and H3.1 as substrate proteins. Samples were analyzed by SDS-PAGE, CBB staining, and Western blotting using the indicated antibodies. (F) Lysates from nocodazole-arrested HeLa cells were used for IP of endogenous cyclin B1, followed by in vitro kinase assays to test phosphorylation of recombinant H2B. Specific antibodies were used to reveal H2B S6ph and precipitation of cyclin B1 and the associated CDK1. The asterisk indicates the position of the light chain from the precipitating antibodies.

Analysis of H2B S6 phosphorylation networks. (A) HeLa cells were transfected with a Dharmacon siRNA library against kinase and kinase-related genes. After 42 h, cells were treated with 200 nM nocodazole for 6 h and prepared for immunofluorescence staining against phosphorylated H2B S6. Phosphorylation intensities were quantified using a wide field Nikon microscope (Eclipse Ti-E) and high-content analysis software. Volcano plot shows standard deviations of average scores from mean H2B S6 phosphorylation intensity (x axis) and their statistical significance (P values; y axis). Statistical analysis was performed using GraphPad Prism. Aurora B, BubR1, and CDK1 are shown in red. (B) GFP-BubR1 and cyclin B1-Venus were expressed in 293T cells in the absence or presence of Myc-INCENP and HA-Aurora B WT or kinase dead (KD). Cells were lysed with SDS sample buffer and analyzed by SDS-PAGE and Western blotting using the indicated antibodies. (C) HeLa cells were arrested at prometaphase with nocodazole for 16 h and released in the presence of MG132 together with various concentrations of the indicated kinase inhibitors. Cells were lysed with SDS sample buffer and further analyzed by immunoblotting as shown. (D) Different plasmids encoding the indicated proteins, including constitutively active (AF) or kinase dead (KD) kinases, were expressed in 293T cells as shown. Aurora B was immunoprecipitated with HA antibodies and CDK1 with anti-Flag M2 affinity gel for a subsequent in vitro kinase assay with recombinant histone octamers as substrate. Samples were analyzed by SDS-PAGE, CBB staining, and immunoblotting using the indicated antibodies; unspecific IgG bands are indicated by asterisks. (E) Cyclin B1-Venus and Flag-tagged CDK1 forms were coexpressed in 293T cells. Cells were lysed with NP-40 buffer, and CDK1 was immunoprecipitated with anti-Flag M2 affinity gel for in vitro kinase assay using H2B and H3.1 as substrate proteins. Samples were analyzed by SDS-PAGE, CBB staining, and Western blotting using the indicated antibodies. (F) Lysates from nocodazole-arrested HeLa cells were used for IP of endogenous cyclin B1, followed by in vitro kinase assays to test phosphorylation of recombinant H2B. Specific antibodies were used to reveal H2B S6ph and precipitation of cyclin B1 and the associated CDK1. The asterisk indicates the position of the light chain from the precipitating antibodies.

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