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. 2020 Feb 7;16(2):e1008597. doi: 10.1371/journal.pgen.1008597

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Fig 4 — (A) Increased stability of endogenous HA-Cse4 but not histone H3 in met30-6 and cdc4-1 strains. Western blot analysis was performed using WCE from WT (YMB9673), cdc4-1 (YMB9571), and met30-6 (YMB8789) strains expressing endogenous HA-Cse4 grown at 25°C. Western blots were probed with anti-HA, anti-H3 and anti-Tub2 (loading control) antibodies. Percentage of remaining HA-Cse4 at 60 minutes after CHX treatment (50 μg/ml) is indicated. Line graphs of the results at different time points is shown on the right. Results from at least two biological experiments are shown as mean ± average deviation. (B) Defects in Cse4 proteolysis in cdc4-1 and met30-6 strains are cell cycle independent. Levels of endogenous HA-Cse4 were analyzed by Western blot analysis as described in (A) except WCE were prepared from cells arrested in either G1 phase (with alpha factor), S phase (with hydroxyurea; HU), or G2/M phase (with nocodazole) for 90 min (S2 Fig). Percentage of remaining HA-Cse4 at 60 minutes after CHX treatment (50 μg/ml) is indicated. Line graphs of the results at different time points are shown on the right. Results from two biological experiments are shown as mean ± average deviation. (C) Stabilized Cse4 is enriched in chromatin. Whole cell extracts, soluble and chromatin fractions from WT (YMB9673), cdc4-1 (YMB9571) and met30-6 (YMB8789) strains expressing endogenous HA-Cse4 grown at 25°C were analyzed by Western blot analysis using anti-HA (HA-Cse4), anti-Tub2, and anti-H3 antibodies. Tub2 and histone H3 were used as markers for soluble and chromatin fractions, respectively. Percentage of HA-Cse4 remaining after 45 minutes of CHX treatment are shown. Results from two biological experiments are shown as mean ± average deviation. (D) Deletion of MET32 does not suppress the defect in Cse4 proteolysis in met30Δ met32Δ strain. Western blot analysis was performed with WCE from WT (YMB9673), met32Δ (YMB10859) and met30Δ met32Δ (YMB10799) strains grown at 25°C. Western blots were probed with anti-HA or anti-Tub2 antibodies. Percentage of HA-Cse4 remaining at 90 minutes after CHX treatment (50 μg/ml) is indicated. Results from two biological experiments are shown as mean ± average deviation. (E) Endogenous HA-Cse4 is stabilized upon depletion of Cdc4. The CDC4 shut-off strain (YMB10212) expressing endogenous HA-Cse4 was grown in galactose at 25°C. CHX (50 ug/ml) treated cells were collected at indicated time points from galactose grown culture (CDC4-ON) or after shift to glucose medium (CDC4-OFF) for 60 min. Western blots were probed with anti-HA or anti-Tub2 (used as a loading control) antibodies. Percentage of HA-Cse4 remaining at 60 minutes after CHX treatment is indicated. Results of at least two biological experiments are shown as mean ± average deviation.