Figure 3. Rad22 protein degradation by Bag101-mediated proteasome.

(a) The amount of Rad22 in cells overexpressing bag101 or containing bag101 deletions. Rad22 was measured with Western blots using anti-Rad22 antibodies. The Rad22 protein levels in no-irradiated cells were represented here and that of the time course after irradiation were shown in Supplementary Figure S2. (b) rad22 mRNA level in cells overexpressing or containing bag101 deletions. The mRNA from each mutant was measured with quantitative PCR using specific rad22 primers. hxk2 was used for internal controls. These experiments were performed three times and the error bars represent the S.D. (c) Physical interaction of the 26S proteasome with Bag101. Pad1 was co-immunoprecipitated with Bag101 in cell lysates expressing Bag101-FH6 and immunoblotted with anti-Pad1 antibody. (d) The stabilization of Rad22 proteins after proteasome inactivation. Rad22 was highly expressed in temperature-sensitive mutants mts2 and mts3 compared to cells expressing wild type mts2 and mts3 after inactivating proteasomes by increasing the temperature from 26°C to 36°C.