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. Author manuscript; available in PMC: 2009 Jul 20.
Published in final edited form as: J Mol Biol. 2007 Jun 4;371(2):514–527. doi: 10.1016/j.jmb.2007.05.095

Figure 3. The degradation of 9-1-1 complex by Jab1 is through proteasome pathway.

Figure 3

Figure 3

(a) Jab1 decreased 9-1-1 protein degradation rate. Myc-tagged Rad1, Hus1 and Rad9 were co-transfected with empty vector or HA-Jab1 expression vector in 293T cells. After 40 hours, cycloheximide (80 μg/mL) was added to the culture, and the whole cell extracts were prepared at 0, 1, 2, 4, and 8 hours and assayed by Western blotting with an anti-Myc antibody to detect 9-1-1 expression levels. (b) The intensity of the bands in (a) was quantitated by phosphorimaging and plotted relative to the amount present at time 0. (c) Proteasome inhibitor increased Jab1-downregulated 9-1-1 protein levels. Myc-tagged Rad1, Hus1 and Rad9 were co-transfected with empty vector or HA-Jab1 expression vector in 293T cells. Cells were incubated with or without the proteasome inhibitor, MG132 (25 μmol/L), for 3 hours. Extracts were assayed by Western blotting with Myc antibody to detect Rad1, Hus1 and Rad9.