Figure 9. Identification of proteins... | Rockefeller University Press

Figure 9.

$Figure 9. Identification of proteins showing S6ph-dependent H2B binding. (A) HeLa cells were arrested in mitosis with nocodazole for 16 h and lysed with NP-40 buffer. The indicated biotin-coupled peptides with unmodified or modified amino acids encompassing the first 20 amino acids from H2B were used for pulldown assays using StrepTactin beads (Qiagen). One fraction of the eluates was analyzed by SDS-PAGE and silver staining. Arrows indicate phosphorylation-dependent interactors. (B) Samples from peptide pulldown experiments were analyzed by SDS-PAGE and Western blotting using SET and 14-3-3 antibodies; arrows show the SET and 14-3-3 isoforms. (C) GFP-tagged H2B WT or S6 phosphorylation site mutants were expressed in 293T cells. Cells were lysed with NP-40 buffer, sonicated, and treated with Benzonase (Millipore). H2B-GFP was immunoprecipitated using the GFP-Trap and SET binding was analyzed by immunoblotting. (D) (His)6-tagged SET (His-SET) and the indicated H2B-GST fusion proteins were purified from E. coli BL21 and then mixed as shown. Following incubation for 2 h of 1 µg GST fusion proteins or 5 µg GST and 0.5 µg his-SET, a GST pulldown was performed and the proteins were detected by CBB staining and immunoblotting as shown. (E) 293T cells were transfected to express GFP, GFP-SET, or GFP-SETΔC which lacks the 59 C-terminal amino acids. The experiment was further performed as in C, with the exception that endogenous H2B was detected by antibodies. (F) SET expression was eliminated in HCT116 cells by CRISPR-Cas9–mediated gene deletion, and a tet-inducible GFP-SET expression plasmid was stably introduced by puromycin selection. Following induction of GFP-SET expression by 1 µg/ml doxycycline for 24 h, cells were arrested with RO-3306 for 16 h and released into mitosis. Cells were stained against H2B S6ph, and the localization of H2B S6ph and GFP-SET in different mitotic phases is displayed. Bars, 2 µm.$

Identification of proteins showing S6ph-dependent H2B binding. (A) HeLa cells were arrested in mitosis with nocodazole for 16 h and lysed with NP-40 buffer. The indicated biotin-coupled peptides with unmodified or modified amino acids encompassing the first 20 amino acids from H2B were used for pulldown assays using StrepTactin beads (Qiagen). One fraction of the eluates was analyzed by SDS-PAGE and silver staining. Arrows indicate phosphorylation-dependent interactors. (B) Samples from peptide pulldown experiments were analyzed by SDS-PAGE and Western blotting using SET and 14-3-3 antibodies; arrows show the SET and 14-3-3 isoforms. (C) GFP-tagged H2B WT or S6 phosphorylation site mutants were expressed in 293T cells. Cells were lysed with NP-40 buffer, sonicated, and treated with Benzonase (Millipore). H2B-GFP was immunoprecipitated using the GFP-Trap and SET binding was analyzed by immunoblotting. (D) (His)6-tagged SET (His-SET) and the indicated H2B-GST fusion proteins were purified from E. coli BL21 and then mixed as shown. Following incubation for 2 h of 1 µg GST fusion proteins or 5 µg GST and 0.5 µg his-SET, a GST pulldown was performed and the proteins were detected by CBB staining and immunoblotting as shown. (E) 293T cells were transfected to express GFP, GFP-SET, or GFP-SETΔC which lacks the 59 C-terminal amino acids. The experiment was further performed as in C, with the exception that endogenous H2B was detected by antibodies. (F) SET expression was eliminated in HCT116 cells by CRISPR-Cas9–mediated gene deletion, and a tet-inducible GFP-SET expression plasmid was stably introduced by puromycin selection. Following induction of GFP-SET expression by 1 µg/ml doxycycline for 24 h, cells were arrested with RO-3306 for 16 h and released into mitosis. Cells were stained against H2B S6ph, and the localization of H2B S6ph and GFP-SET in different mitotic phases is displayed. Bars, 2 µm.