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. 2015 Oct 17;43(20):9889–9904. doi: 10.1093/nar/gkv1064

Figure 4.

Figure 4.

HERC3 interacts with RelA and mediates its ubiquitination. (A) RelA was precipitated with RelA antibody from total HEK293T cell lysates transfected with myc-RelA and myc-HERC3. RelA and HERC3 co-precipitation was assessed by Western Blotting with anti-myc antibody. (B) HEK293T cells were transfected with HA-RelA and myc-HERC3. HERC3 was pulled down from cell lysates with myc-specific antibody and RelA was detected in the precipitate with anti-RelA antibody. (C) HEK293T were transfected with myc-RelA, his-ubiquitin and myc-HERC3. Ubiquitinated RelA was detected by pull down of his-ubiquitin under denaturing conditions, followed by Western Blotting with RelA-specific antibody. (D) BAEC were transfected with his-ubiquitin and myc-HERC3. After 24 h cells were treated with TNF for 30 min and assay was performed as described in (C). All experiments were carried out in triplicates. IB, immunoblot; IP, immunoprecipitation.