Identification of mitotic H2B S6ph. (A) The sites of the H2B N-terminal tail known to be modified by phosphorylation (ph), acetylation (ac), methylation (me), or ubiquination (ub) are shown in black; the novel phosphorylation of S6 is marked in red. (B) 293T cells were treated with 50 nM calyculin A for 30 min, and lysates were incubated with λ phosphatase as shown. Equal amounts of protein contained in cell lysates were analyzed by Western blotting for the occurrence and phosphorylation of H2B with specific antibodies. The positions of molecular weight markers are indicated. (C) HeLa cells were treated with the indicated reagents or the DMSO vehicle control for 16 h and lysed with SDS sample buffer. Lysates were analyzed by SDS-PAGE and Western blotting using the indicated antibodies; the cleaved form of the caspase substrate PARP is indicated by an asterisk. (D) HeLa cells were synchronized by a double thymidine block and released into S phase. Cells were lysed at the indicated time points with SDS sample buffer. Lysates were analyzed by SDS-PAGE and Western blotting using the indicated antibodies. (E) Diploid RPE-1 cells were fixed, and immunostaining was performed with antibodies against H2B S6ph and α-tubulin. DNA was visualized with Hoechst 33342. Bars: 5 µm (main); 2 µm (magnification). 3D-SIM was used to reveal the occurrence and localization of the stained proteins during the indicated mitotic phases; the boxed areas are displayed in 4× magnification. (F) HeLa cells were synchronized by treatment with nocodazole for 16 h and released in the presence of 10 µM MG132 or DMSO. Cells were lysed at the indicated time points with SDS sample buffer and analyzed by SDS-PAGE and Western blotting using the indicated antibodies.