Fig 1: Lys29-linkage of ASK1 is crucial for innate antiviral response.(A) Map3k5+/+ RAW264.7 cells and Map3k5–/– RAW264.7 cells transfected with mock vector or indicated ASK1 vectors for 48 hr were examined for ASK1 expression by Western blot. (B–D) Map3k5+/+ and Map3k5–/– RAW264.7 cells (as in A) were infected with or without (Mock) VSV (MOI = 1) or HSV-1 (MOI = 5) as indicated for 8 hr (B, C) or 12 hr (D). IL-6 (B) and IFNβ (C) concentrations in supernatant were determined by ELISA. The titers of viruses in cells were determined by plaque formation assay (D). Error bars indicated for mean ± SD of triplicate samples. Data were analyzed by one-way ANOVA followed by Bonferroni multiple comparison using PRISM software (ns, not significant; ***p < 0.001). (EG) Cells in (A) were infected with or without (Mock) VSV (MOI = 1) or HSV-1 (MOI = 5) as indicated. Then whole cell lysate (E, F) or immunoprecipitates (G) were examined by Western blot. In (G), the immunoprecipitated ASK1 was examined for the kinase activity in the presence of 10 ng recombinant MKK4. One representative experiment of three was shown.DOI: http://dx.doi.org/10.7554/eLife.14087.018
Fig 2: Lys29-linkage of ASK1 is crucial for Fbxo21-mediated ASK1 activation.(A) HEK293 cells transiently transfected with indicated vectors for 48 hr and infected with VSV (MOI = 1) for 2 hr in the presence or absence of MLN-4924 (10 nM). Then ASK1 (mutant) was immunoprecipitated (IP) with anti-Flag agaroses, and polyubiquitination was evaluated by immunblot (IB) against HA. (B) Fbxo21–/– RAW264.7 cells were transfected with indicated vectors for 48 hr and infected with VSV (MOI = 1) or HSV-1 (MOI = 5) for 2 hr. Then ASK1 (mutant) was immunoprecipitated (IP) with anti-Flag agaroses, and polyubiquitination was evaluated by immunblot (IB) against Ub. (C, D) Fbxo21-/–- RAW264.7 cells were transfected with indicated vectors for 48 hr and infected with VSV (MOI = 1, C) or HSV-1 (MOI = 5, D) for 2 hr. Then ASK1 (mutant) was immunoprecipitated (IP) with anti-Flag agaroses, and the phosphorylated ASK1 was examined by immunoblot. Otherwise, the ASK1 (mutant) after IP was incubated with recombinant MKK4, and then immunoblotted for phosphorylated MKK4. One representative experiment of three was shown.DOI: http://dx.doi.org/10.7554/eLife.14087.014
Fig 3: Fbxo21 interacts with ASK1.(A, B) Wild type RAW264.7 cells or L929 cells infected with (B) or without (A) VSV and HSV-1 were prepared for whole cell extracts (WCE), which were immunoprecipitated (IP) with anti-Fbxo21 or IgG as indicated. The associated ASK1 was examined by immunoblot. (C) Schema of ASK1 mutants. CC1, coiled-coil domain 1; KD, kinase domain; CC2, coiled-coil domain 2. (D) HEK293 cells were cotransfected with Fbxo21-Myc and indicated Flag-tagged ASK1 mutants (1–4 mutants as illustrated in (C)) for 48 hr. Whole cell lysates immunoprecipitated (IP) with anti-Myc agaroses were examined for Flag-tagged ASK1 (mutant) by immunoblot. (E) Schema of Fbxo21 mutants. F-box, F-box domain; CC, coiled-coil domain; YccV, YccV domain. (F) HEK293 cells were cotransfected with ASK1-Flag and indicated Myc-tagged Fbxo21 mutants (1–4 mutants as illustrated in (E)) for 48 hr. Whole cell lysates immunoprecipitated (IP) with anti-Flag agaroses were examined for Myc-tagged Fbxo21 (mutant) by immunoblot. (G) GST pull-down assays of Fbxo21 interaction with recombinant (Rec) ASK1. '0' indicates for GST alone, and 1–4 indicate for GST-fused Fbxo21 (mutant) as in (E). One representative experiment of three was shown.DOI: http://dx.doi.org/10.7554/eLife.14087.009
Fig 4: SCFFbxo21 mediates virus-induced Lys29-linkage of ASK1.(A) Fbxo21+/+ and Fbxo21-/- RAW264.7 cells were infected with VSV (MOI=1) or HSV-1 (MOI = 5) for 2 hr. Then polyubiquitination of ASK1 was examined by immunoblot (IB) after immunoprecipitations (IP). (B) Fbxo21+/+ and Fbxo21-/- RAW264.7 cells were infected with VSV (MOI = 1) or HSV-1 (MOI = 5) as indicated. ASK1 and Fbxo21 levels in whole cell lysates were examined by immunoblot. (C) Myc-tagged Fbxo21 and Flag-tagged ASK1 were cotransfected with HA-tagged Ub (mutant) into HEK293 cells for 48 hr, and infected with VSV (MOI = 1) for 2 hr. Then polyubiquitinated ASK1 was examined by immunoblot (IB) against HA after immuneprecipitations (IP) with anti-Flag agaroses. 1, wild type Ub; 2, Ub with only the Lys6 residue unchanged (K6O-Ub); 3, K11O-Ub; 4, K27O-Ub; 5, K29O-Ub; 6, K33O-Ub; 7, K48O-Ub; 8, K63O-Ub. (D, E) Fbxo21-/- RAW264.7 cells were transfected with indicated amounts (0, 1 or 5 μg) Fbxo21-Myc and equal amounts of K29O-Ub-HA vectors for 48 hr, and then infected with VSV (MOI = 1) or HSV-1 (MOI = 5) for 2 hr in the presence or absence of MLN-4924 (10 nM). Then polyubiquitinated ASK1 was examined by immunoblot (IB) against HA after immuneprecipitations (IP). (F) After incubation for 30 min, the in vitro polyubiquitination system was boiled for 5 min, and then polyubiquitinated ASK1 was examined by immunoblot (IB) against Ub after immuneprecipitations (IP). One representative experiment of three was shown.DOI: http://dx.doi.org/10.7554/eLife.14087.012
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