Figure 5.
Figure 5. ASC is linear ubiquitinated by LUBAC in 293T cells and in vitro. (a) Flag-tagged ASC, AIM2, or NLRP3 were cotransfected with HOIL-1L-HA and myc-HOIP into 293T cells. Linear ubiquitinated proteins were immunoprecipitated. WCL (whole cell lysate) or immunoprecipitates (IP Lin Ub) were immunoblotted for Flag and linear ubiquitin. (b) 293T cells were transfected with a panel of WT HOIL-1L-HA and Flag-HOIP or RBR E3 ligase domain cysteine to serine point mutants (cs) and probed by linear ubiquitin IP for ASC-V5, HA, and Flag as indicated. (c) Transfected 293T cells were treated for 6 h with 20 µM MG132 or an equivalent volume of DMSO before lysates were collected and immunoprecipitated with linear ubiquitin antibody. Immunoprecipitates were probed for linear ubiquitin, Flag, and ubiquitin as indicated. (d) Bacterially purified GST-CoZi wt and rre proteins were incubated with lysates from 293T cells transfected with ASC-Flag, HOIL-1L-HA, and myc-HOIP or vector control plasmids (left, input). Proteins that were pulled down by WT or rre mutant GST-CoZi were probed for linear ubiquitin or Flag by immunoblotting (right, GST-Cozi PD). (e) ASC-Flag and NLRP3-Flag were purified from 293T lysates in a nondenaturing high salt buffer (500 mM NaCl) and combined as indicated with E1 (Ube1), E2 (UbcH5c), and LUBAC (HOIL-1L/HOIP) purified from bacteria in a linear ubiquitination reaction that was incubated for 18 h at 37°C. Reactions were stopped by the addition of loading dye and immunoblotted for Flag. Membranes were then stripped and reprobed for linear ubiquitin. (f) Deubiquitinase treatment of linear ubiquitin immunoprecipitates from 293T cell lysates overexpressing HOIL-1L-HA, myc-HOIP, and ASC-V5. Reactions were immunoblotted for linear ubiquitin and V5. Control purified ubiquitin chains were incubated in parallel in deubiquitinase reactions that were immunoblotted with a linkage nonspecific antibody against ubiquitin (Ub). All data are representative of a minimum of three independent experiments. * indicates unmodified size. Protein size markers are indicated in kD.

ASC is linear ubiquitinated by LUBAC in 293T cells and in vitro. (a) Flag-tagged ASC, AIM2, or NLRP3 were cotransfected with HOIL-1L-HA and myc-HOIP into 293T cells. Linear ubiquitinated proteins were immunoprecipitated. WCL (whole cell lysate) or immunoprecipitates (IP Lin Ub) were immunoblotted for Flag and linear ubiquitin. (b) 293T cells were transfected with a panel of WT HOIL-1L-HA and Flag-HOIP or RBR E3 ligase domain cysteine to serine point mutants (cs) and probed by linear ubiquitin IP for ASC-V5, HA, and Flag as indicated. (c) Transfected 293T cells were treated for 6 h with 20 µM MG132 or an equivalent volume of DMSO before lysates were collected and immunoprecipitated with linear ubiquitin antibody. Immunoprecipitates were probed for linear ubiquitin, Flag, and ubiquitin as indicated. (d) Bacterially purified GST-CoZi wt and rre proteins were incubated with lysates from 293T cells transfected with ASC-Flag, HOIL-1L-HA, and myc-HOIP or vector control plasmids (left, input). Proteins that were pulled down by WT or rre mutant GST-CoZi were probed for linear ubiquitin or Flag by immunoblotting (right, GST-Cozi PD). (e) ASC-Flag and NLRP3-Flag were purified from 293T lysates in a nondenaturing high salt buffer (500 mM NaCl) and combined as indicated with E1 (Ube1), E2 (UbcH5c), and LUBAC (HOIL-1L/HOIP) purified from bacteria in a linear ubiquitination reaction that was incubated for 18 h at 37°C. Reactions were stopped by the addition of loading dye and immunoblotted for Flag. Membranes were then stripped and reprobed for linear ubiquitin. (f) Deubiquitinase treatment of linear ubiquitin immunoprecipitates from 293T cell lysates overexpressing HOIL-1L-HA, myc-HOIP, and ASC-V5. Reactions were immunoblotted for linear ubiquitin and V5. Control purified ubiquitin chains were incubated in parallel in deubiquitinase reactions that were immunoblotted with a linkage nonspecific antibody against ubiquitin (Ub). All data are representative of a minimum of three independent experiments. * indicates unmodified size. Protein size markers are indicated in kD.

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