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. 2006 May 11;25(11):2420–2431. doi: 10.1038/sj.emboj.7601110

Figure 3.

Figure 3

Cyh sensitivity and turnover of Rhp6-dependent ubiquitination of H2B in htb1 mutants. (A) The htb1 mutants are hypersensitive to Cyh, a protein synthesis inhibitor. Both htb1-72 and htb1-223 were slower in growth than wild type in the presence of Cyh. Cells were serially diluted (1:5) and spotted onto the YPD plates containing Cyh (0, 2 and 5 μg/ml). The highest-density spots contained 5 × 104 cells. (B) The cell number increase (left panel) and the frequencies of unequal nuclear division (right) are shown for wild type (open) and htb1-223 (closed) in the absence (circles) or presence (rectangles, triangles) of Cyh at 33°C. (C) Immunoblot patterns using polyclonal antibodies against histone H2B (anti-H2B) and monoclonal antibodies against HA (anti-HA). Extracts of cells expressing endogenous H2B (Rep1 vector) and ectopically expressed H2B tagged with HA (pRep1-htb1HA) in the presence of thiamine (promoter off) and absence of thiamine (14 h after removal of thiamine; promoter on) were used for immunoblot. The 17 kDa H2B band and the 24–25 kDa uH2B bands (indicated by the arrows) were detected in cells carrying vector. In cells carrying pRep1-htb1HA, additional bands for H2B-3HA (asterisks) were detected by anti-H2B and anti-HA antibodies. (D) Immunoblot was performed for extracts of wild type, Δrhp6 carrying vector or plasmid pRHP6. The presumed monoubiquitinated H2B band was missing in Δrhp6. (E) Wild type, htb1-72 and htb1-223 were cultured in the absence (−) or presence of (+) of Cyh (100 μg/ml) at 36°C for 0–180 min. In the case of +Cyh, strains were first cultured at 36°C for 60 min and then followed by the addition of Cyh. Immunoblot patterns by short and long exposure are shown. The positions for H2B and uH2B are indicated by arrowheads. (F) Wild type, mts2-1, htb1-223 and the double mutant mts2-1 htb1-223 were cultured first at 26°C, and then shifted to 36°C for 8 h. Immunoblot patterns using anti-H2B antibodies indicated that the level of decrease of uH2B was not arrested in the proteasome mutant mts2-1 background.