Transfected ABIN-2-FL coimmunoprecipitates with both HA-p105 and Myc-TPL-2. (A and B) 293 cells were cotransfected with vectors encoding HA-p105, HA-p50, or Myc-TPL-2; with empty vector (EV) and ABIN-2-FL; or with EV alone as indicated. Anti-HA (α-HA), anti-Myc, and anti-FL immunoprecipitates (Ips) and cell lysates were Western blotted with the indicated antibodies. (C) 293 cells were cotransfected with vectors encoding HA-p105 or with EV and ABIN-2-FL. Cell lysates were cleared of endogenous TPL-2 by immunoprecipitation with anti-TPL-2 antibody and then reimmunoprecipitated with anti-HA MAb. Immunoprecipitates and lysates were Western blotted with the indicated antibodies. (D) 293 cells were cotransfected with the indicated expression vectors. Cell lysates were cleared of endogenous p105 by immunoprecipitation with anti-p105C antibody and then reimmunoprecipitated with anti-FL MAb to isolate ABIN-2-FL and associated Myc-TPL-2. Immunoprecipitates and lysates were Western blotted with the indicated antibodies.

The majority of endogenous ABIN-2 forms a ternary complex with p105 and TPL-2. (A) BMDM lysate was immunoprecipitated with anti-ABIN-2 antibody (α-ABIN-2) (Ip Ab) or control preimmune rabbit IgG (PI). Isolated protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. (B to D) ABIN-2, p105, and TPL-2 were individually removed from lysates of BMDMs by immunoprecipitation with specific antibodies. Control preclearing was performed using preimmune rabbit IgG (PI). Precleared lysates were then reimmunoprecipitated (Re-Ip) with the indicated specific antibodies. Reimmunoprecipitated protein (B and D) and precleared cell lysate (C) was resolved by SDS-PAGE (10% acrylamide) and Western blotting.

Mapping regions of ABIN-2 which interact with p105 and TPL-2. (A) Schematic diagram of recombinant GST-ABIN-2 fusion proteins. The positions of the ABIN homology domain (AHD) and the binding regions for TPL-2, p105, and A20 are shown. The N and C termini of the wild-type (WT) GST-ABIN-2 protein (amino acids 1 to 420) are indicated. (B and C) 293 cells were transfected with vectors encoding Myc-p105, Myc-TPL-2, or Myc-A20. Cell lysates were prepared using 1% Brij 58 buffer A and incubated with the indicated GST-ABIN-2 fusion proteins or GST (control) coupled to glutathione-Sepharose 4B. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.

nf-κb1^−/− cells have dramatically reduced levels of ABIN-2 protein. (A and C) Lysates from nf-κb1^−/− and wild-type (nf-κb1^+/+) 3T3 fibroblasts (A) and BMDMs (C) were immunoprecipitated with the indicated antibodies. Immunoprecipitates (Ip) and cell lysates were resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti. (B) ABIN-2 mRNA levels in total RNA isolated from nf-κb1^−/− and wild-type 3T3 fibroblasts were assayed by semiquantitative PCR. The 18S rRNA amplicon was used as an internal control.

ABIN-2 copurifies with NF-κB1 p105. (A) Protein was purified from lysate of HeLa S3 cells stably transfected with HA-p105(S927A) or empty vector (EV) by sequential affinity purification using anti-HA MAb and anti-p105C antibody. Purified protein was resolved by SDS-PAGE (10% acrylamide) and revealed by silver staining. The positions of the identified proteins are shown. For mass spectroscopic analysis, the indicated 100-kDa regions (bands 1 and 2) and 50-kDa regions (bands 3 to 8) were excised as a series of adjacent slices numbered from high to low molecular weight from a replicate SDS-polyacrylamide gel (10% acrylamide) stained with colloidal Coomassie brilliant blue (not shown). Proteins in isolated bands were identified by MALDI mass spectroscopy of tryptic digests. (B) ABIN-2 amino acid sequence showing the deduced portions of peptides (in bold type) identified by MALDI mass spectroscopic analysis of band 7. Peptide coverage corresponded to 56% of the ABIN-2 amino acid sequence. (C) Lysates of HeLa S3 cells were immunoprecipitated with the indicated specific antibodies (IP Ab) or preimmune (PI) IgG. Isolated proteins were resolved by SDS-PAGE (10% acrylamide) and Western blotting. The specificity of blotting and immunoprecipitating antibodies is denoted as α (anti) followed by the name of the recognized antigen.

ABIN-2 preferentially interacts with a p105/TPL-2 complex. (A and B) Duplicate cultures of 293 cells were cotransfected with vectors encoding ABIN2-FL and HA-p105 or Myc-TPL-2 or with EV. Cell lysates were prepared from each duplicate culture set using either buffer A (1% NP-40) or RIPA buffer, as indicated. Lysates were resolved by SDS-PAGE (10% acrylamide) and Western blotting (top blots). HA-p105 and Myc-TPL-2 mRNA levels in total RNA were assayed by semiquantitative RT-PCR (bottom blots). The 18S rRNA amplicon was used as an internal control. (C) 293 cells were cotransfected with vectors encoding HA-p105 and TPL-2 individually or together. Transfected proteins were affinity purified from cell lysates, prepared in 1% NP-40 buffer A, using GST-ABIN-2_1-429 fusion protein coupled to glutathione-Sepharose. Isolated proteins were resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.

Mapping interacting regions for ABIN-2 on p105 and TPL-2. (A) Schematic diagram of HA-p105 mutants. The relative positions of the Rel homology domain (RHD), ankyrin repeats (ANK), death domain (DD), and PEST region are shown. The N and C termini of the wild-type (WT) HA-p105 protein (amino acids 1 to 968) are indicated. (B) 293 cells were transfected with vectors encoding wild-type (WT) and mutant forms of HA-p105. Cell lysates, prepared using 1% Brij 58 buffer A, were incubated with GST-ABIN-2_1-429 fusion protein or GST (control) coupled to glutathione-Sepharose beads. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti. (C) GST-p105_497-968 fusion protein and GST (control) were coupled to glutathione-Sepharose beads and used to affinity purify ABIN-2-FL translated and labeled with [³⁵S]methionine in vitro. Isolated protein was detected by autoradiography of SDS-8% acrylamide gels. (D) 293 cells were transfected with vectors encoding wild-type and mutant forms of Myc-TPL-2. GST-ABIN-2_1-429 was used as an affinity ligand to isolate protein from cell lysates prepared with 1% NP-40 buffer A. Isolated protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. (E) TPL-2_398-467 peptide coupled to streptavidin-agarose beads was used as an affinity ligand to isolate ABIN-2-FL from lysates of transfected 293 cells. Bound protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting.

Depletion of ABIN-2 by RNA interference dramatically reduces steady-state levels of TPL-2. (A and B) ABIN-2 expression in HeLa S3 cells (A) and 293 cells (B) was decreased by siRNA treatment (−ABIN-2). Control cells were treated with an irrelevant siRNA oligonucleotide pair (+ABIN-2). Cell lysates were resolved by SDS-PAGE (10% acrylamide) and Western blotting (top blots). TPL-2 mRNA levels in total RNA were assayed by semiquantitative RT-PCR (bottom blots). The 18S rRNA amplicon was used as an internal control. α, anti. (C) ABIN-2 was depleted by RNA interference in HeLa and 293 cells as described above for panels A and B. Expression of p105, p50, or ABIN-2 was determined by Western blotting of cell lysates. The position of the ABIN-2 band in the Western blot of HeLa cell lysates is indicated by an asterisk. (D) 293 cells were cotransfected with vectors encoding TPL-2 and ABIN-2-FL or with EV. Lysates were resolved by SDS-PAGE (10% acrylamide) and Western blotting (top blots). In duplicate cultures, transfected TPL-2 mRNA levels were determined by semiquantitative RT-PCR (bottom blots). 18S rRNA was used as an internal control. (E) 293 cells were cotransfected with expression vectors encoding TPL-2 and ABIN-2-FL or with EV. After 24 h, cells were metabolically pulse-labeled with [³⁵S]methionine-[³⁵S]cysteine (30 min) and then chased for the times indicated. Anti-TPL-2 immunoprecipitates were resolved by SDS-PAGE (8% acrylamide) and visualized by fluorography. Amounts of immunoprecipitated labeled TPL-2 were quantified by densitometry (n = 3).

ABIN-2 is not associated with active TPL-2 in LPS-stimulated BMDMs. (A and B) BMDMs from wild-type BALB/C mice were stimulated for the indicated times with LPS or left unstimulated. In panel B, BMDMs were preincubated with MG132 or vehicle control for 30 min prior to stimulation with LPS. Lysates were resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti. (C) BMDMs were stimulated for 15 min with LPS or left unstimulated. Lysates were immunoprecipitated (Ip) with the indicated antibodies, and associated MEK kinase activity was determined by coupled MEK/ERK kinase assay (KA). Labeled MBP was visualized by autoradiography after SDS-PAGE (10% acrylamide). Levels of immunoprecipitated proteins were determined by Western blotting. The LPS-induced mobility shift of M1-TPL-2 is due to phosphorylation (data not shown). (D) BMDM lysates were depleted of ABIN-2 by immunoprecipitation with anti-ABIN-2 antibody. ABIN-2 precleared lysates and control untreated lysates were then Western blotted. (E) 293 cells were cotransfected with vectors encoding Myc-TPL-2 and ABIN-2-FL or with EV. The MEK kinase activity of immunoprecipitated Myc-TPL-2 was determined using GST-MEK1(K207A) protein as a substrate. Phosphorylation was assayed by Western blotting of reaction mixtures and probing with anti-phospho-MEK-1/2 antibody (α-P-MEK). Western blotting of anti-Myc immunoprecipitates demonstrated that similar amounts of TPL-2 were assayed in each reaction mixture.