Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 6

1.
Figure 3

Figure 3. TRE17 co-immunoprecipitates with IKK. From: Atypical Mechanism of NF-?B activation by TRE17/Ubiquitin-Specific Protease 6 (USP6) oncogene and its requirement in tumorigenesis.

(A) MC3T3 cell lines expressing vector, TRE17(long) (denoted T17 or TRE17(long)), or TRE17(long)/USP- (USP-) were treated with dox as indicated. Cell extracts were immunoprecipitated with anti-IKKα or non-immune (n.i.) antibody, then blotted back for TRE17, IKKα, and IKKβ. WCL, whole cell lysate. (B) HeLa cells were transfected with HA- or GST-tagged TRE17(long), then immunoprecipitated with IKKα (α) or non-immune (ni) antibody. Samples were blotted for TRE17, IKKα, IKKβ, and NEMO. (C) Control (vec) or HA-TRE17(long)-expressing MC3T3 cell lines were subjected to immunoprecipitation using anti-HA antibody, then blotted back for IKKα and IKKβ.

Lashon M. Pringle, et al. Oncogene. ;31(30):3525-3535.
2.
Figure 6

Figure 6. TRE17-induced tumorigenesis is dependent on USP activity and NFκB. From: Atypical Mechanism of NF-?B activation by TRE17/Ubiquitin-Specific Protease 6 (USP6) oncogene and its requirement in tumorigenesis.

(A) Nude mice were injected subcutaneously with stable NIH3T3 cell lines expressing vector, TRE17(long), or TRE17(long)/USP-. Animals are shown 11 days post-injection. (B) Stable NIH3T3 cell lines expressing TRE17(long) alone or together with IκBSR were treated with dox, then subjected to immunoblotting with the indicated antibodies. (C) EMSA was performed using the indicated cell lines, treated with dox or TNFα (Tα) as shown. (D) Nude mice were subcutaneously injected with TRE17(long) (top) or TRE17(long)-IκBSR cells (bottom). (E) Extensive tumor vascularization was observed in mice injected with TRE17(long)/NIH3T3 but not TRE17(long)-IκBSR/NIH3T3.

Lashon M. Pringle, et al. Oncogene. ;31(30):3525-3535.
3.
Figure 4

Figure 4. IKKα and IKKβ are both required for optimal activation of NFκB by TRE17. From: Atypical Mechanism of NF-?B activation by TRE17/Ubiquitin-Specific Protease 6 (USP6) oncogene and its requirement in tumorigenesis.

(A) HeLa cells were subjected to two rounds of transfection with SmartPool siRNAs targeting IKKα (α), IKKβ (β), NEMO (N), or a control non-targeting sequence (C). Whole cell lysates were blotted as indicated to confirm specific knockdown of the different IKK subunits. (B) HeLa cells treated with the indicated siRNA were transfected with HA-TRE17(long) or vector, and vector cells were treated with TNFα (Tα) where indicated. Samples were subjected to cell fractionation; cytosolic fractions were blotted for TRE17 to confirm uniform expression (top). Nuclear fractions were subjected to EMSA (middle), or blotted for p65 (bottom). HDAC2 was monitored as a loading control.

Lashon M. Pringle, et al. Oncogene. ;31(30):3525-3535.
4.
Figure 1

Figure 1. TRE17 activates the canonical NFκB pathway. From: Atypical Mechanism of NF-?B activation by TRE17/Ubiquitin-Specific Protease 6 (USP6) oncogene and its requirement in tumorigenesis.

(A) HeLa cells were transiently transfected with HA-TRE17(long), NIK, or control vector, and treated with TNFα (10 ng/ml for 30 minutes) where indicated. Nuclear extracts were prepared and subjected to EMSA. Unlabelled oligonucleotide (cold oligo) and antibodies (Ab) against the specific NFκB subunits were added where indicated. RB, RelB. (B) Stable MC3T3 cell lines expressing HA-TRE17(long), a USP-inactive point mutant (USP-), or control vector in a doxycyclin (dox)-inducible manner were subjected to immunoblotting with TRE17 antibody (left). Nuclear extracts were prepared from the cell lines treated as specified, then subjected to EMSA/supershift assays (right). (C) Stable NIH3T3 cell lines expressing HA-TRE17(long), TRE17(long)/USP-, or control vector were blotted with TRE17 antibody (left). EMSA was performed using equal amounts of nuclear extract from the indicated cell line (middle). Supershift assays were performed using the indicated antibodies (right). (D) HeLa cells were transfected with GFP-TRE17(long) or a constitutively active phospho-mimetic mutant of IKKα, then starved. Samples were subjected to immunofluorescence confocal microscopy, using antibodies for p65 and p100/p52. Arrowheads indicate transfected cells.

Lashon M. Pringle, et al. Oncogene. ;31(30):3525-3535.
5.
Figure 5

Figure 5. TRE17 stimulates IKK activity and p65 S536 phosphorylation in a manner dependent on both IKKα and IKKβ. From: Atypical Mechanism of NF-?B activation by TRE17/Ubiquitin-Specific Protease 6 (USP6) oncogene and its requirement in tumorigenesis.

(A) MC3T3 cells expressing control vector, TRE17(long) (T17(long)), or TRE17(long)/USP- (USP-) were treated with dox or TNFα. Extracts were immunoprecipitated with anti-IKKα or non-immune (n.i.) antibody, then subjected to in vitro kinase assays using recombinant GST-p65 (top) or GST- I B (bottom) as a substrate. Half of the reaction was subjected to autoradiography (left panels). The other half was blotted for anti-IKKα to confirm uniform immunoprecipitation; whole cell extracts were blotted with TRE17 (right panels). (B) MC3T3 cell lines were treated with or without dox, and extracts were blotted as indicated. (C) HeLa cells were with the indicated siRNA (IKKα, α; IKKβ, β; NEMO, N; and control, C). Samples were then transfected with vector or HA-TRE17(long), and treated with TNFα. Extracts were blotted as indicated. (D) HeLa cells were transfected with siRNA against TRE17 (T) or control siRNA (C), then stimulated with TNFα or LIGHT (100 ng/ml). Extracts were blotted as shown. (E and F) Nuclear extracts were prepared from HeLa cells transfected with TRE17 (T) or control (C) siRNA, then subjected to EMSA (E) or blotting for p65 and HDAC2 (F).

Lashon M. Pringle, et al. Oncogene. ;31(30):3525-3535.
6.
Figure 2

Figure 2. TRE17 activates classical NFκB independently of IκB phosphorylation and degradation. From: Atypical Mechanism of NF-?B activation by TRE17/Ubiquitin-Specific Protease 6 (USP6) oncogene and its requirement in tumorigenesis.

(A) MC3T3 cell lines were treated with dox as indicated. Whole cell extracts (left) or anti-p65 immunoprecipitates were subjected to immunoblotting with the indicated antibodies. (B) TRE17(long)/MC3T3 cells were treated with dox for various times, then subjected to immunoblotting with the indicated antibodies. HeLa extracts were probed with IκBα and phospho-IκBα (right). Open arrowheads indicate migration of IκBα and phospho-IκBα; black arrowheads indicate non-specific crossreactive band. (C) Control or TRE17(long)MC3T3 cell lines were treated with cycloheximide (CHX; 25 μg/ml) for the indicated times, in the absence or presence of TNFα as indicated. Extracts were blotted with the indicated antibody. Note that in cells treated with TNFα for 2 hr in the absence of CHX (lane 9), IκBβ re-synthesis starts to occur. (D) MC3T3 cell lines were treated as indicated, and subjected to cell fractionation. Nuclear extracts were immunoprecipitated with anti-p65, then blotted for IkB proteins. HDAC2 blotting of total nuclear extracts confirmed that equal amounts of protein were used. (E) Control or TRE17(long)-expressing (T17) MC3T3 cells were treated with dox or TNFα (Tα), then immunoprecipitated simultaneously with anti-IκBα and -IκBβ. Supernatants were blotted directly, or subjected to a second round of IκB immunoprecipitation. Both IκB α and IκBβ were efficiently immunodepleted after the first round of immunoprecipitation (note that IκBβ was monitored by blotting extracts after immunodepletion rather than directly in the immunoprecipitates, since it co-migrated with rabbit IgG).

Lashon M. Pringle, et al. Oncogene. ;31(30):3525-3535.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is temporarily unavailable.

Write to the Help Desk