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1.
Figure 7

Figure 7. From: The kinase IKK? inhibits activation of the transcription factor NF-?B by phosphorylating the regulatory molecule TAX1BP1.

Phospho-mimetic TAX1BP1 bypasses the requirement for IKKα in terminating NF-κB signaling. Immunoblot analysis of lysates of Tax1bp1−/− MEFs transfected with empty vector or expression vector for Flag-tagged wild-type TAX1BP1, TAX1BP1(S593A, S624A) or TAX1BP1(S593E, S624E), together with control siRNA or IKKα-specific siRNA, then treated for 0–30 min with TNF (a) or IL-1 (b); blots were probed with anti-Flag, anti-IκBα, anti-IKKα or anti-β-actin. Data are representative of two experiments.

Noula Shembade, et al. Nat Immunol. ;12(9):834-843.
2.
Figure 4

Figure 4. From: The kinase IKK? inhibits activation of the transcription factor NF-?B by phosphorylating the regulatory molecule TAX1BP1.

Phosphorylation of TAX1BP1 regulates recruitment of the A20 ubiquitin-editing complex to TRAF2 and TRAF6. Immunoassay of lysates of Tax1bp1−/− MEFs transfected with empty vector or expression vector for Flag-tagged TAX1BP1 or TAX1BP1(S593A, S624A), then treated for 0–120 min with TNF (a) or IL-1 (b), assessed after immunoprecipitation with anti-TRAF2 (a) or anti-TRAF6 (b) by immunoblot analysis with antibodies along left margins (a, b). Below (Lysates), immunoblot analysis of total cell lysates with antibodies along left margins. Data are representative of at least three experiments.

Noula Shembade, et al. Nat Immunol. ;12(9):834-843.
3.
Figure 3

Figure 3. From: The kinase IKK? inhibits activation of the transcription factor NF-?B by phosphorylating the regulatory molecule TAX1BP1.

Phosphorylation of TAX1BP1 is essential for the termination of NF-κB signaling, Jnk phosphorylation and RIP1 ubiquitination. (a, b) Immunoassay of lysates of Tax1bp1−/− MEFs transfected with empty vector or expression vector for Flag-tagged wild-type TAX1BP1 or Flag-TAX1BP1(S593A, S624A), then treated for 0–120 min with TNF (a) or IL-1 (b), assessed after immunoprecipitation with anti-RIP1 by immunoblot analysis with antibody specific for K63-linked ubiquitin (K63-Ub) and anti-RIP1 (a) or after immunoprecipitation with anti-TRAF6 by immunoblot analysis with antibody specific for K63-linked ubiquitin and anti-TRAF6 (b). Below (Lysates), immunoblot analysis of total cell lysates with antibodies along left margin. (c) Immunoblot analysis of lysates of mouse BMDMs transfected with siRNA specific for mouse Tax1bp1, together with empty vector or expression vector for Flag-tagged wild-type human TAX1BP1 or TAX1BP1(S593A, S624A), and treated for 0–60 min with TNF, probed with anti-TAX1BP1, anti-IκBα or anti-β-actin. Data are representative of at least three experiments.

Noula Shembade, et al. Nat Immunol. ;12(9):834-843.
4.
Figure 5

Figure 5. From: The kinase IKK? inhibits activation of the transcription factor NF-?B by phosphorylating the regulatory molecule TAX1BP1.

Phosphorylation of TAX1BP1 is required for assembly of the A20 ubiquitin-editing complex. (a, b) NF-κB luciferase activity in lysates of Tax1bp1−/− MEFs transfected with an NF-κB firefly luciferase reporter and a renilla luciferase vector reporter, plus empty vector or expression vector for Flag-tagged wild-type TAX1BP1, TAX1BP1(S593A), TAX1BP1(S624A) or TAX1BP1(S593A, S624A), then treated for 8 h with TNF (a) or IL-1 (b); results are presented relative to renilla luciferase activity. Below, immunoblot analysis of the cells above with anti-Flag. (c, d) Immunoassay of lysates of Tax1bp1−/− MEFs transfected with empty vector or expression vector for Flag-tagged wild-type TAX1BP1, TAX1BP1(S593A), TAX1BP1(S624A) or TAX1BP1(S593A, S624A), then treated for 0–120 min with TNF (c) or IL-1 (d), assessed after immunoprecipitation with anti-RNF11 by immunoblot analysis with antibodies along left margins. Below (Lysates), immunoblot analysis of total cell lysates with antibodies along left margins. Data are representative of at least three experiments (error bars (a, b), s.e.m. of triplicates).

Noula Shembade, et al. Nat Immunol. ;12(9):834-843.
5.
Figure 6

Figure 6. From: The kinase IKK? inhibits activation of the transcription factor NF-?B by phosphorylating the regulatory molecule TAX1BP1.

IKKα is essential for assembly of the A20 ubiquitin-editing complex and termination of NF-κB signaling. (a) Immunoassay of lysates of wild-type, Ikka−/−, Ikbkb−/− and Ikbkg−/− MEFs, left untreated or treated for 15 min with TNF, assessed after immunoprecipitation with anti-RIP1 or antibody to isotype-matched control immunoglobulin (α-Ig) by immunoblot analysis with anti-A20 or anti-RIP1. (b) Immunoassay of lysates of wild-type and Ikka−/− MEFs treated as in a, assessed after immunoprecipitation with anti-TAX1BP1 or control antibody (as in a) by immunoblot analysis with anti-Itch or anti-TAX1BP1. Below (Lysates; a, b), immunoblot analysis of total cell lysates with anti-IκBα and anti-β-actin. (c, d) Immunoassay of lysates of wild-type MEFs transfected with control siRNA (Ctrl) or IKKα-specific siRNA and treated with TNF (c) or IL-1 (d), assessed after immunoprecipitation with anti-A20 or anti-TAX1BP1 by immunoblot analysis with antibodies along left margins. Anti-IκBα, anti-IKKα or anti-β-actin performed on total cell lysates (c, d). (e, f) Immunoassay of lysates of mouse BMDMs treated for 0–120 min with TNF (e) or IL-1 (f), assessed after immunoprecipitation with anti-RIP1 by immunoblot analysis with antibody specific for K63-linked ubiquitin and anti-RIP1 (e) or after immunoprecipitation with anti-TRAF6 by immunoblot analysis with antibody specific for K63-linked ubiquitin or anti-TRAF6 (f). Below (Lysates), immunoblot analysis of total cell lysates with anti-IKKα, anti-IκBα or anti-β-actin. Ig HC (f), immunoglobulin heavy chain. Data are representative of at least three experiments.

Noula Shembade, et al. Nat Immunol. ;12(9):834-843.
6.
Figure 8

Figure 8. From: The kinase IKK? inhibits activation of the transcription factor NF-?B by phosphorylating the regulatory molecule TAX1BP1.

HTLV-I Tax blocks the phosphorylation of TAX1BP1. (a) Immunoblot analysis of lysates of wild-type MEFs transfected with expression vector for Flag-tagged TAX1BP1 plus plasmid encoding Tax, then treated with TNF or IL-1, probed with anti-Flag, anti-β-actin plus anti-IκBα or anti-Tax. (b) Immunoblot analysis of lysates of wild-type MEFs transfected with empty plasmid or plasmid encoding Tax, then treated for 0–120 min with IL-1 or TNF, probed with antibody specific for TAX1BP1 phosphorylated at Ser593, anti-TAX1BP1, anti-β-actin plus anti-IκBα or anti-Tax. (c) Immunoassay of lysates of wild-type MEFs transfected with empty plasmid or plasmid encoding Tax, then treated for 30 min with TNF or IL-1 (above lanes), assessed after immunoprecipitation with anti-TAX1BP1 by immunoblot analysis with anti-IKKα or anti-TAX1BP1. Below (Lysates), immunoblot analysis of total cell lysates with anti-IκBα, anti-Tax or anti-β-actin. (d) Immunoblot analysis of lysates of the adult T cell leukemia cell lines TL-OM1, MT-2 and C8166 and of Jurkat Tax Tet-on cells left untreated (−) or treated (+) for 15 min with PMA and ionomycin (P-I) with (+) or without (−) treatment for 16 h with doxycycline (Dox; 1 μg/ml); blots were probed with antibody specific for TAX1BP1 phosphorylated at Ser593, anti-TAX1BP1, anti-Tax, antibody to phosphorylated IκBα or anti-β-actin. (e) NF-κB luciferase assay of lysates of Tax1bp1−/− MEFs transfected with an NF-κB firefly luciferase reporter and a renilla luciferase vector reporter, plus empty vector or expression vector for Flag-tagged wild-type TAX1BP1 or TAX1BP1(S593E, S624E) (EE), with (Tax) or without (−) plasmid encoding Tax; results are presented relative to renilla luciferase activity. Below, immunoblot analysis with anti-TAX1BP1 or anti-Tax. Data are representative of at least three experiments (error bars (e), s.e.m. of triplicates).

Noula Shembade, et al. Nat Immunol. ;12(9):834-843.
7.
Figure 1

Figure 1. From: The kinase IKK? inhibits activation of the transcription factor NF-?B by phosphorylating the regulatory molecule TAX1BP1.

Phosphorylation of TAX1BP1 in response to proinflammatory stimuli. (a) Immunoblot analysis (IB) of lysates of MEFs transfected with expression vector for Flag-tagged TAX1BP1 (Flag-TAX1BP1), then left untreated (NT) or treated for 30 min with TNF or LPS (10 ng/ml). α-, anti-; p-, phosphorylated. (b) Immunoblot analysis of lysates of MEFs treated with calf intestinal alkaline phosphatase (CIP) and left unstimulated (−) or stimulated for 30 min (+) with TNF (10 ng/ml). (c) In vivo kinase assay of MEFs labeled with 32P-orthophosphate and left untreated (−) or treated for 15 min (+) with TNF (10 ng/ml), followed by immunoprecipitation of proteins from lysates with anti-TAX1BP1 and autoradiography for visualization of phosphorylated TAX1BP1. kDa, kilodaltons. (d) Immunoblot analysis of lysates of 293T cells transfected with expression vector for Flag-TAX1BP1 and left untransfected (−) or transfected with expression vector for hemagglutinin (HA)-tagged IKKα, IKKβ or IKKγ (above lanes). (e) Immunoblot analysis of lysates of wild-type (WT), Ikka−/−, Ikbkb−/− or Ikbkg−/− MEFs left untransfected (−) or transfected (+) with expression vector for Flag-TAX1BP1 and left untreated (−) or treated (+) for 15 min with TNF. Anti-β-actin serves as a loading control throughout. (f) In vitro kinase assay of recombinant active IKKα or IKKβ, with a fusion of GST and TAX1BP1 amino acids 205–599 (GST-TAX1BP1(205–599)) or IκBα amino acids 1–54 (GST-IκBα (1–54)) as the substrate. Far left (−), GST fusion proteins alone; bottom blots in each, immunoblot analysis of GST fusion proteins with anti-GST. (g) Immunoassay of TAX1BP1 interactions in Ikka−/− MEFs transfected with expression vectors for HA-IKKα or HA-IKKα (K44M) and left untreated or treated for 15 min with TNF or IL-1, detected in lysates after immunoprecipitation (IP) with anti-TAX1BP1 (top two blots) by immunoblot analysis with anti-HA or anti-TAX1BP1; below, immunoblot analysis of total cell lysates with anti-HA, anti-IκBα or anti-β-actin. (h) Immunoblot analysis of lysates of Ikka−/− MEFs transfected with empty vector (EV) or expression vectors for Flag-TAX1BP1, plus HA-IKKα or HA-IKKα (K44M), then left untreated (−) or treated (+) for 15 min with TNF (left) or IL-1 (right); blots were probed with anti-Flag, anti-β-actin, anti-IκBα or anti-IKKα. Data are representative of at least three experiments.

Noula Shembade, et al. Nat Immunol. ;12(9):834-843.
8.
Figure 2

Figure 2. From: The kinase IKK? inhibits activation of the transcription factor NF-?B by phosphorylating the regulatory molecule TAX1BP1.

Phosphorylation of TAX1BP1 on Ser593 and Ser624 in response to stimulation with TNF or IL-1. (a) Consensus IKK-phosphorylation sites in IκBα (Ser32 and Ser36, S) and putative IKK-phosphorylation sites in TAX1BP1 (Ser212, Ser254 and Ser593, S). Top, consensus phosphorylation sites, with phosphorylated serine (pS) at position 0 and acidic amino acids aspartic acid (D) upstream at position −5 and glutamic acid (E) downstream at position +3. pT, phosphorylated threonine; L/I, leucine or isoleucine; pT/D, phosphorylated threonine or aspartic acid; Y, tyrosine; X, any amino acid. (b) TAX1BP1 deletion mutants (left): SKICH, SKIP (skeletal muscle and kidney enriched inositol phosphatase) carboxyl homology domain; CC, coiled coil domain; ZnF, zinc-finger domain; far left, amino acids. Right, phosphorylation results. (c) Immunoblot analysis of lysates of 293T cells transfected with expression vector for Flag-tagged TAX1BP1 deletion mutants in b, with or without HA-tagged IKKα (above lanes). (d) Silver-stained gel of 293T cells transfected with expression vector for Flag-TAX1BP1 and left untreated or treated for 30 min with TNF (top), and identification of phosphorylated peptides in those cells by matrix-assisted laser desorption-ionization quadrupole ion–trap time-of-flight (bottom): far left, observed mass; right, amino acid sequence (phosphorylated residues underlined). (e) Immunoblot analysis of lysates of 293T cells transfected with empty vector or expression vector for wild-type TAX1BP1 or TAX1BP1 (S593A), with or without IKKα (above lanes). (f) Immunoblot analysis of lysates of Tax1bp1−/− MEFs transfected with expression vector for wild-type or mutant TAX1BP1 (below blots) and left untreated or treated with TNF or IL-1 (above lanes); blots were probed with anti-Flag, anti-β-actin or anti-IκBα. (g) Sequence alignment of the phosphorylation site of TAX1BP1 (S; human Ser624) in various species. (h) In vivo kinase assay of Tax1bp1−/− MEFs reconstituted with empty vector or expression vector for Flag-tagged wild-type TAX1BP1 or TAX1BP1(S593A, S624A) (AA), labeled with 32P-orthophosphate and left untreated or treated for 15 min with TNF (10 ng/ml), followed by immunoprecipitation and autoradiography (as in Fig. 1c); blots were probed with anti-TAX1BP1 (after immunoprecipitation with anti-TAX1BP1) or anti-β-actin and anti-IκBα (total lysates). (i) Immunoblot analysis of lysates of 293T cells transfected with various expression vectors (above lanes), probed with antibody specific for TAX1BP1 phosphorylated at Ser593 (α-p-TAX1BP1), anti-Flag or anti-HA. (j) Immunoblot analysis of lysates of Tax1bp1+/− and Tax1bp1−/− MEFs treated for 0–120 min with TNF, probed with the phosphorylation-specific antibody in i, anti-IκBα or anti-β-actin. (k) Immunoblot analysis of lysates of mouse BMDMs treated for 0–60 min with TNF or IL-1, probed with the phosphorylation-specific antibody in j, anti-TAX1BP1, anti-IκBα or anti-β-actin. Data are representative of at least three experiments.

Noula Shembade, et al. Nat Immunol. ;12(9):834-843.

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