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. 2018 Feb 15;7(3):bio031575. doi: 10.1242/bio.031575

Fig. 2.

Fig. 2.

ZYG11B-based E3 ligase supports co-precipitation of polyubiquitin with ZFP36L2. (A) ZFP36L2 is down-regulated in interphase cells by protein degradation. HeLa cells expressing Flag-tagged ZFP36L2 were synchronized either at M phase (thymidine-nocodazole block) or interphase (serum-free cultivation), and then treated for an additional 4 h with (+) or without (−) 10 µM MG-132. Densitometry quantification of Flag immunoblot signals relative to loading control is shown below each lane. (B) Co-immunoprecipitation (IP) experiments to detect polyubiquitin association of ZFP36L2. Flag-ZFP36L2 and T7-tagged ubiquitin (T7-Ub) were co-expressed in HeLa cells, and the cells were treated with (+) or without (−) 10 µM MG-132 for 4 h. Anti-Flag immunoprecipitates from the protein lysates were probed with an anti-T7 antibody to detect polyubiquitin co-precipitation of ZFP36L2. Note that ZFP36L2 loading was adjusted. Densitometry quantification of T7 immunoblot signals relative to Flag signals is shown below each lane. (C) ZFP36L2 physically interacts with ZYG11B protein. Flag-tagged ZFP36L2 was co-expressed in HeLa cells with T7-tagged ZYG11B. An anti-Flag M2 antibody was used for immunoprecipitation. (D) Interaction of ZFP36L2 with ZYG11B was enhanced in interphase cells. HeLa cells expressing Flag-tagged ZFP36L2 and T7-ZYG11B were harvested either at interphase (In) or M phase (M), and then ZFP36L2 was immunoprecipitated using an anti-Flag M2 antibody and probed with an anti-T7 antibody. Densitometry quantification of T7 immunoblot signals relative to Flag signals is shown below each lane. (E-H) ZYG11B and CUL2 knockdown weakened the co-precipitation of polyubiquitin with ZFP36L2 protein. Flag-tagged ZFP36L2 and T7-Ub were expressed in siRNA-treated HCT116 cells with MG-132 (E,G). Flag precipitates were probed with an anti-T7 antibody to detect the co-precipitation of polyubiquitin with ZFP36L2. Graphs indicate the quantified data of the polyubiquitin blot signals that were co-immunoprecipitated with ZFP36L2 protein from ZYG11B knockdown cells (F) and CUL2 knockdown cells (H). ZYG11B knockdown experiments were replicated independently three times, and CUL2 knockdown experiments were replicated twice. The efficacy of ZYG11B and CUL2 siRNA knockdown was verified by western blot analysis (see also Fig. S3A,B).