Figure 5. 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

Figure 5—figure supplement 1. MS assays of Fbxo21-associated proteins.

Wild type RAW264.7 cells were treated with or without 100 ng/ml LPS or VSV (MOI = 1) as indicated. Whole cells lysates were immunoprecipitated (IP) with anti-Fbxo21 antibody plus protein A/G beads. The immune complexes were separated in a SDS-PAGE gel, and stained with silver buffer. The differential bands were subjected to MS assays. The identified unique peptides of ASK1 were shown at the right side.

DOI: http://dx.doi.org/10.7554/eLife.14087.010

Figure 5—figure supplement 2. Fbxo21 is a component of SCF complex.

(A) Wild type RAW264.7 cells were treated with or without VSV (MOI = 1) or HSV-1 (MOI = 5) as indicated for 2 hr. Whole cell lysates immunoprecipitated (IP) with anti-Fbxo21 antibody plus protein A/G agaroses were examined for indicated SCF components by immunoblot (IB). (B–D) HEK293 cells were cotransfected with Myc-tagged Fbxo21 or Fbxo21 without F-box domain (#2) and indicated Flag-tagged SCF components for 48 hr. Whole cell lysates immunoprecipitated (IP) with anti-Myc agaroses were examined for indicated Flag-tagged proteins by immunoblot (IB). One representative experiment of three was shown.

DOI: http://dx.doi.org/10.7554/eLife.14087.011