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In an effort to investigate how loss of deneddylation affects SCF activity, …


Biology Articles » Biochemistry » Protein Biochemistry » Targeted silencing of Jab1/Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels » Figures

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- Targeted silencing of Jab1/Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels

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Figure 1. Suppression of Csn5 in HEK 293 human cells. (A) Expression of Csn5-specific shRNA suppresses endogenous Csn5 mRNA levels. Cells induced with doxycycline for 8 days were harvested and analyzed by RT-PCR using oligos specific for Csn5. (B) Expression of Csn5-specific shRNA suppresses endogenous Csn5 protein levels. Cells induced with doxycycline for 8 days were harvested and lysed. Lysates were analyzed by western blot using antibodies specific toward human Csn5. (C) Silencing of Csn5 does not appreciably affect the levels of other CSN subunits. Cells induced with doxycycline for 8 days were harvested, and cell lysates were analyzed by western with the antibodies indicated. (D) Silencing of Csn5 results in altered cullin neddylation. Cells were induced with doxycycline for 8 days and harvested. Cells were lysed and analyzed by western blot using the antibodies indicated.

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Figure 2. Loss of Csn5 results in a decrease in F-box protein levels. (A) Depletion of Csn5 results in a decrease in F-box protein levels. Cells were treated with doxycycline for eight days followed by lysis and SDS-PAGE/western blot analysis with antibodies toward the proteins indicated. (B) Depletion of Csn5 does not affect F-box protein mRNA levels. Cells were treated with doxycycline for eight days and analyzed by RT-PCT using oligos specific toward the mRNAs indicated.

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Figure 3. Loss of Csn5 results in proteasome-dependent turnover of F-box proteins. (A) Supplementation of doxycycline-treated cells with MG132 inhibits the turnover of F-box proteins. Cells were treated with doxycycline for eight days followed by treatment with MG132 (25 μM) for eight hours. Cells were lysed in SDS and analyzed by SDS-PAGE/western blot for the indicated proteins. (B) Ectopic expression of wild-type, but not JAMM point mutant Csn5, can rescue the decrease in protein levels of Skp2 and cyclin F. Cells were treated with doxycycline for six days, followed by transient transfection with wild-type Flag-Csn5, Flag-Csn5 (ASA), or Flag-Csn5 (D151N). Forty-eight hours post-transfection, cells were lysed in SDS and analyzed by SDS-PAGE/western blot for the indicated proteins. (C) Ectopic expression of a dominant-negative Cul1 restores cyclin F protein levels. Control, wild-type, or Cul1 (1–428) expression plasmids were transfected into cells induced with doxycycline for six days. Forty-eight hours post-transfection, cells were lysed in SDS and analyzed by SDS-PAGE/western blot with antibodies against the indicated proteins.

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Figure 4. Loss of Csn5 results in an increase in cyclin E, an F-box protein substrate. (A) Substrates of the Fbw7 SCF complex accumulate when Csn5 is depleted. Cells were induced with doxycycline for eight days followed by SDS-PAGE/western blot analysis for the indicated proteins. (B) Activity of the protein kinase cyclin E/Cdk2 is increased when cells are depleted for Csn5. Cells were induced with doxycycline for eight days followed by lysis under native conditions. Lysates were immunoprecipitated with antibodies toward cyclin E and immuoprecipitates analyzed for kinase activity against histone H1. (C) Cell cycle regulation of cyclin E is lost when cells are depleted for Csn5. Cells were induced with doxycycline for eight days followed by arrest at the indicated cell cycle stages. Cell lysates were analyzed by SDS-PAGE/western blot with the indicated antibodies.

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