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Fig. 2. TSA treatment decreases the Mis4 protein level and the chromatin-bound portion of Rad21 in the chromosome arm region. (A) Wild-type and mis4-242 cells were cultured in YPD medium at 26°C and were harvested at the indicated time points after TSA (12.5 µg/ml) was added. Immunoblotting for the cell extracts was carried out for Mis4, Rad21, Cut9, Cdc2 (PSTAIR) and acetylated histone H3 and H4 (AcH3 and AcH4, respectively). (B) Non-tagged wild type (WT No tag), rad21HA integrant strains (WT rad21HA) and cut9-665rad21HA strains were cultured at 26°C, and then incubated at 36°C for 4 hours with or without TSA (12.5 µg/ml). ChIP for Rad21HA was performed using anti-HA antibody followed by PCR with four sets of primers (cnt1 and dg for the central and outer centromere, respectively, and lys1 and c83 for the chromosome arm region). Immunoblotting for the whole cell extracts of the unfixed cells was also carried out using the indicated antibodies (WB). (C) Non-tagged wild-type (No tag) and rad21HA strains were grown exponentially at 26°C and cells were fixed at the indicated time points after TSA (12.5 µg/ml) was added to the cultures. ChIP was carried out as B using seven sets of primers (imr1 for the central centromere on chromosome I, 08c and ARS2004 for the arm).
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