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. 2019 Feb 26;41:538–555. doi: 10.1016/j.ebiom.2019.02.009

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© 2019 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Fig. 4 — PLEKHO1 interacts with TRAF2 to promote the TRAF2-mediated ubiquitination of RIP1. (a) Levels of inflammatory cytokines (IL-1β, IL-6) produced by MC3T3E1 cells transfected with Plekho1 siRNA or random siRNA and subjected to TNF-α (10 ng/ml). (b) MC3T3E1 cells transfected with Plekho1 siRNA or random siRNA were stimulated with TNF-α (10 ng/ml) for 45 min, and harvested for detection of IKKα/β and p65 phosphorylation. (c) MC3T3E1 cells transfected with Plekho1 siRNA or random siRNA were stimulated with TNF-α (10 ng/ml) for 45 min, and harvested for detection of RIP1 ubiquitination. (d) Quantification of relative level of RIP1 ubiquitination. (e, f) Immunoprecipitation using anti-Flag antibody to examine the PLEKHO1-TRAF2 interaction and mapping of the PLEKHO1-interacting domain in TRAF2. e, The Flag-tagged full-length TRAF2 (Flag-TRAF2) and various truncated mutants (Flag-ΔRing, Flag-ΔRing-Zinc, Flag-TRAF N and Flag-ΔTRAF C) were shown with amino acid numbers. f, HEK293T cells were transfected with Myc-PLEKHO1 and Flag-TRAF2 or truncated mutants as indicated. Full-length TRAF2 or truncated mutants was immunoprecipitated by anti-Flag antibody. PLEKHO1 in immunoprecipitates or whole-cell lysate was detected by anti-Myc antibody. (g, h) Immunoprecipitation using anti-Myc antibody to examine the PLEKHO1-TRAF2 interaction and mapping of the TRAF2-interacting domain in PLEKHO1. g, The Myc-tagged full-length PLEKHO1 (Myc-PLEKHO1) and various truncated mutants (Myc-ΔPH, Myc-LZ, and Myc-ΔLZ) were shown with amino acid numbers. h, HEK293T cells were transfected with Flag-TRAF2 and Myc-PLEKHO1 or truncated mutants as indicated. Full-length PLEKHO1 or truncated mutants was immunoprecipitated by anti-Myc antibody. TRAF2 in immunoprecipitates or whole-cell lysate was detected by anti-Flag antibody. (i) In vivo ubiquitination assay of RIP1 in HEK293T cells transfected with Myc-RIP1, HA-Ubiquitin (HA-Ub) and Flag-TRAF2, along with Flag-PLEKHO1 or Flag-ΔLZ. Ubiquitination of RIP1 was detected in Myc immunoprecipitates. (j) Quantification of relative level of RIP1 ubiquitination. (k) In vivo ubiquitination assay of RIP1 in HEK293T cells transfected with Myc-RIP1, HA-Ub and Flag-PLEKHO1, along with Flag-TRAF2 or Flag-ΔTRAF C. Ubiquitination of RIP1 was detected in Myc immunoprecipitates. (l) Quantification of relative level of RIP1 ubiquitination. Note: IP, Immunoprecipitation; IB, immunoblotting. Data are representative of three independent experiments. **P < 0.01. Comparisons between two groups were performed using a Student's t-test.

Fig. 4 — PLEKHO1 interacts with TRAF2 to promote the TRAF2-mediated ubiquitination of RIP1. (a) Levels of inflammatory cytokines (IL-1β, IL-6) produced by MC3T3E1 cells transfected with Plekho1 siRNA or random siRNA and subjected to TNF-α (10 ng/ml). (b) MC3T3E1 cells transfected with Plekho1 siRNA or random siRNA were stimulated with TNF-α (10 ng/ml) for 45 min, and harvested for detection of IKKα/β and p65 phosphorylation. (c) MC3T3E1 cells transfected with Plekho1 siRNA or random siRNA were stimulated with TNF-α (10 ng/ml) for 45 min, and harvested for detection of RIP1 ubiquitination. (d) Quantification of relative level of RIP1 ubiquitination. (e, f) Immunoprecipitation using anti-Flag antibody to examine the PLEKHO1-TRAF2 interaction and mapping of the PLEKHO1-interacting domain in TRAF2. e, The Flag-tagged full-length TRAF2 (Flag-TRAF2) and various truncated mutants (Flag-ΔRing, Flag-ΔRing-Zinc, Flag-TRAF N and Flag-ΔTRAF C) were shown with amino acid numbers. f, HEK293T cells were transfected with Myc-PLEKHO1 and Flag-TRAF2 or truncated mutants as indicated. Full-length TRAF2 or truncated mutants was immunoprecipitated by anti-Flag antibody. PLEKHO1 in immunoprecipitates or whole-cell lysate was detected by anti-Myc antibody. (g, h) Immunoprecipitation using anti-Myc antibody to examine the PLEKHO1-TRAF2 interaction and mapping of the TRAF2-interacting domain in PLEKHO1. g, The Myc-tagged full-length PLEKHO1 (Myc-PLEKHO1) and various truncated mutants (Myc-ΔPH, Myc-LZ, and Myc-ΔLZ) were shown with amino acid numbers. h, HEK293T cells were transfected with Flag-TRAF2 and Myc-PLEKHO1 or truncated mutants as indicated. Full-length PLEKHO1 or truncated mutants was immunoprecipitated by anti-Myc antibody. TRAF2 in immunoprecipitates or whole-cell lysate was detected by anti-Flag antibody. (i) In vivo ubiquitination assay of RIP1 in HEK293T cells transfected with Myc-RIP1, HA-Ubiquitin (HA-Ub) and Flag-TRAF2, along with Flag-PLEKHO1 or Flag-ΔLZ. Ubiquitination of RIP1 was detected in Myc immunoprecipitates. (j) Quantification of relative level of RIP1 ubiquitination. (k) In vivo ubiquitination assay of RIP1 in HEK293T cells transfected with Myc-RIP1, HA-Ub and Flag-PLEKHO1, along with Flag-TRAF2 or Flag-ΔTRAF C. Ubiquitination of RIP1 was detected in Myc immunoprecipitates. (l) Quantification of relative level of RIP1 ubiquitination. Note: IP, Immunoprecipitation; IB, immunoblotting. Data are representative of three independent experiments. **P < 0.01. Comparisons between two groups were performed using a Student's t-test.