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. Author manuscript; available in PMC: 2022 Jan 25.
Published in final edited form as: Cell Rep. 2021 Dec 28;37(13):110175. doi: 10.1016/j.celrep.2021.110175

Figure 3. LGP2 interferes with TRAF6 Ub ligase independent of TRAF6 interaction.

Figure 3.

(A) Diagrammatic representation of full-length (FL) TRAF6 and truncated variants, illustrating positions of RING domain, Zn finger, TRAF-N, and TRAF-C domains and highlighting the 8-amino-acid (8aa) difference critical for LGP2 co-precipitation.

(B) HEK293T cells were transfected with expression vectors for HA-tagged LGP2 protein along with the FLAG-tagged TRAF6 proteins, and lysates were subjected to FLAG M2 immunoaffinity purification and immunoblotting to detect LGP2 or TRAF6 proteins in both the lysate and the immunoprecipitate (IP).

(C) HEK293T cells were transfected with (+) FLAG-tagged TRAF6, 351 (amino acids 1–351) and 359 (amino acids 1–359), HA-tagged ubiquitin, and 6His-tagged LGP2 as indicated. Lysates were subjected to FLAG immunoaffinity purification and blotting with anti-HA for ubiquitin, anti-LGP2 for LGP2, anti-FLAG for TRAF6, 351, and 359, and anti-GAPDH as a loading control. For all panels, representative experiments are shown of n ≥ 3 replicates.

(D) HEK293T cells were transfected with (+) FLAG-tagged TRAF6, 351 (amino acids 1–351) and 359 (amino acids 1–359), HA-tagged K63-only Ub, and 6His-tagged LGP2 as indicated. Lysates were subjected to FLAG immunoaffinity purification and blotting with anti-HA for K63-only ubiquitin, antiLGP2 for LGP2, anti-FLAG for TRAF6, 351, and 359, and anti-GAPDH as a loading control. For all panels, representative experiments are shown of n ≥ 3 replicates.

(E–G) NF-κB luciferase reporter gene assays with (+) or without (−) expression of FLAG-tagged TRAF6 (E), 351 (F), or 359 (G) with or without 4 ng, 20 ng, 80 ng, or 400 ng of HA-tagged LGP2 vector. Cells were harvested at 24 h post transfection for luciferase assays. Bars represent average values (n = 3) ± standard deviation. Corresponding immunoblots are shown in Figure S5. ***p < 0.0005 by two-tailed Student’s t test.