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Results: 6

1.
Figure 6

Figure 6. Working model. From: TAK1 ubiquitination regulates Doxorubicin-induced NF-?B activation.

Dox-induced double-stranded breaks (DSBs) activate PARP-1 and ATM. PARP-1 signalosome formation promotes NEMO SUMOylation. The coupled export of ATM and sumoylated NEMO translocates to the cytoplasm and assembles a large complex containing TRAF6, cIAP, ELKS. Lys63-linked polyubiquitination of TRAF6 or ELKS recruits TAB2/TAK1 complex and TRAF6 further promotes Lys63-linked polyubiquitination of TAK1 at Lys-158 site. TAK1 with Lys63-linked polyubiquitination recruits IKKs complex and activates IKKs and NF-κB. After TAK1 activation, USP4 deubiquitinates TAK1 with Lys63-linked polyubiquitination and inhibits TAK1-mediated downstream signal transduction. Meanwhile, E3 ligase ITCH catalyzes Lys48-linked polyubiquitination of TAK1 and promotes TAK1 degradation to terminate downstream signal transduction.

Li Liang, et al. Cell Signal. ;25(1):247-254.
2.
Figure 1

Figure 1. TAK1 is required for doxorubicin (Dox) induced NF-κB, p38 and JNK activation. From: TAK1 ubiquitination regulates Doxorubicin-induced NF-?B activation.

(A) TAK1+/+ and TAK1−/− MEFs were treated with Dox at the indicated time points or left untreated, protein extracts were subjected to SDS-PAGE and immunoblotted with antibodies indicated. (B) TAK1-deficient and TAK1 wildtype reconstituted MEF cells were treated with Dox at the indicated time points or left untreated, protein extracts were subjected to SDS-PAGE and immunoblotted with antibodies indicated. (C) TAK1+/+ and TAK1−/− MEFs were treated with VP-16 at the indicated time points or left untreated, protein extracts were subjected to SDS-PAGE and immunoblotted with antibodies indicated. (D) TAK1+/+ and TAK1−/− MEFs were treated with CPT at the indicated time points or left untreated, protein extracts were subjected to SDS-PAGE and immunoblotted with antibodies indicated. (E) TAK1+/+ and TAK1−/− MEFs were treated with ionizing radiation (IR) at the indicated dose or left untreated, protein extracts were subjected to SDS-PAGE and immunoblotted with antibodies indicated. β-actin was detected as a loading control for whole cell extracts.

Li Liang, et al. Cell Signal. ;25(1):247-254.
3.
Figure 3

Figure 3. USP4 deubiquitinates Lys63-linked TAK1 polyubiquitination and inhibits Dox-induced NF-κB activation. From: TAK1 ubiquitination regulates Doxorubicin-induced NF-?B activation.

(A) Expression vectors encoding HA-Ub-K63 and FLAG-TAK1 were co-transfected into HEK-293T cells with control vector or expression vectors encoding MYC-USP4. Thirty six hours after transfection, cells were treated with Dox at the indicated time points or left untreated. FLAG-TAK1 proteins in the transfected cells were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-HA antibodies to detect the presence of polyubiquitinated FLAG-TAK1. (B) USP4 stable knockdown and sh-Control HeLa cells were treated with Dox at the indicated time points or left untreated. Then, protein extracts were subjected to SDS-PAGE and immunoblotted with antibodies indicated.

Li Liang, et al. Cell Signal. ;25(1):247-254.
4.
Figure 4

Figure 4. Dox induces Lys48-linked TAK1 polyubiquitination and degradation. From: TAK1 ubiquitination regulates Doxorubicin-induced NF-?B activation.

(A) Expression vectors encoding HA-Ub-K48 and FLAG-TAK1 were co-transfected into HEK-293T cells. Thirty six hours after transfection, cells were treated with Dox and MG132 at the indicated time points or left untreated. FLAG-TAK1 proteins in the cell lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-HA antibodies to detect the presence of polyubiquitinated FLAG-TAK1. (B) Stable expression of FLAG-TAK1 or control HeLa cells were treated with Dox and MG132 at the indicated time points or left untreated. FLAG-TAK1 proteins in the cell lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-ubiquitin antibodies to detect the presence of ubiquitinated FLAG-TAK1. (C) HeLa cells were treated with Dox and cycloheximide (CHX) at the indicated time points or left untreated. TAK1 proteins in the cell lysates were detected by anti-TAK1 antibodies. (D) Quantification of C.

Li Liang, et al. Cell Signal. ;25(1):247-254.
5.
Figure 5

Figure 5. ITCH promotes Lys48-linked TAK1 polyubiquitination and inhibits Dox-induced NF-κB activation. From: TAK1 ubiquitination regulates Doxorubicin-induced NF-?B activation.

(A) Expression vectors encoding HA-Ub-WT, HA-Ub-K63, HA-Ub-K48 and TAK1-V5His were co-transfected into HEK-293T cells with control vector or expression vectors encoding FLAG-ITCH. Thirty six hours after transfection, TAK1-V5His proteins in the cell lysates were immunoprecipitated with anti-V5 antibodies and immunoblotted with anti-HA antibodies to detect the presence of polyubiquitinated TAK1-V5His. (B) Expression vectors encoding HA-Ub-K48 and TAK1-wildtype-V5His or TAK1-K72R-V5His were co-transfected into HEK-293T cells with control vector or expression vectors encoding FLAG-ITCH. Thirty six hours after transfection, TAK1-V5His proteins in the cell lysates were immunoprecipitated with anti-V5 antibodies and immunoblotted with anti-HA antibodies to detect the presence of polyubiquitinated TAK1-V5His. (C) ITCH+/+ and ITCH−/− MEFs were treated with CHX and Dox at the indicated time points or left untreated, protein extracts were subjected to SDS-PAGE and immunoblotted with antibodies indicated. (D) ITCH+/+ and ITCH−/− MEFs were treated with Dox at the indicated time points or left untreated, protein extracts were subjected to SDS-PAGE and immunoblotted with antibodies indicated.

Li Liang, et al. Cell Signal. ;25(1):247-254.
6.
Figure 2

Figure 2. Lys63-linked TAK1 polyubiquitination at Lys-158 is required for Dox-induced NF-κB activation. From: TAK1 ubiquitination regulates Doxorubicin-induced NF-?B activation.

(A) Expression vectors encoding HA-Ub-K63 and FLAG-TAK1 were co-transfected into HEK-293T cells. Thirty six hours after transfection, cells were treated with Dox at the indicated time points or left untreated. FLAG-TAK1 proteins in the cell lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-HA antibodies to detect the presence of polyubiquitinated FLAG-TAK1. (B) HeLa cells with stable expression of FLAG-TAK1 were treated with Dox at the indicated time points or left untreated. Then, FLAG-TAK1 proteins in the cell lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-ubiquitin antibodies to detect the presence of polyubiquitinated FLAG-TAK1. (C) TAK1 wildtype and TAK1 K158R mutant reconstituted MEF cells were treated with Dox at the indicated time points or left untreated, cell extracts were subjected to SDS-PAGE and immunoblotted with antibodies indicated. (D) pBabe vector, TAK1 wildtype and TAK1 K158R mutant reconstituted TAK1-deficient MEFs were seeded into 96-well plates at the concentration of 10,000 cells per well. 24 hours later, cells were treated by Dox at the indicated concentrations for 48 hours. Cell viability was measured by the CCK-8 assay following the manufacturer’s instructions.

Li Liang, et al. Cell Signal. ;25(1):247-254.

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