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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 2. Jab1 destabilizes the 9-1-1 complex.

Figure 2

(a) Jab1 downregulates overexpressed 9-1-1 in 293T cells. Myc-tagged Rad1, Hus1 and Rad9 were cotransfected with either empty vector (lane 1) or HA-tagged Jab1 in 293T cells. Forty hours after transfection, cell lysates were prepared and subjected to SDS-PAGE/immunoblot analysis with anti-Myc, anti-HA or anti-β-actin antibodies. (b) Jab1 downregulates endogenous 9-1-1 in PANC-1, MiaPaCa-2 and HeLa cells. Cells were infected with retrovirus containing pMSCVneo-GFP or pMSCVneo-HA-Jab1. Cell extracts were analyzed by immunoblotting using antibodies against Rad1, Hus1, Rad9, HA and β-actin. (c) siGFP efficiently eliminated GFP expression in PANC-1 cells. PANC-1 were infected by retrovirus containing pMSCVneo/U6 (empty vector, i) or pMSCVneo/U6/GFP (siGFP, ii), and GFP expression plasmid was transfected in these cells. Green light representing GFP expression. (d) siJab1 elevated 9-1-1 protein levels. PANC-1 cells were infected with virus containing siGFP or three siJab1s individually. Cells were harvested and expression levels of Jab1, Rad1, Hus1, Rad9 and β-actin were measured by Western blot analysis.