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. 2020 Feb 10;295(14):4696–4708. doi: 10.1074/jbc.RA119.011280

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© 2020 Ullah et al.

Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

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Figure 3. — BMAL1 interacts with the STUB1 U-box domain and STUB1 interacts with the BMAL1 middle domain. A, illustration of domains for the GST-tagged full-length STUB1 and its truncations. B, BMAL1 preferably interacts with the STUB1 U-box domain. GST-tagged STUB1 and its truncations were expressed in E. coli, purified with GSH-agarose beads, incubated with cell lysate from HEK293T cells expressing FLAG-BMAL1, washed, and eluted by heating with 2× SDS loading buffer. The eluates were stained with Ponceau S or immunoblotted (WB) with an anti-FLAG antibody. C, illustration of domains for the FLAG-tagged full-length BMAL1 and its truncations. D, STUB1 interacts with the BMAL1 middle domain. FLAG-tagged full-length BMAL1 and its truncations were co-transfected with Myc-STUB1 into HEK293T cells. Cells were lysed, and BMAL1 and its truncations were immunoprecipitated with an anti-FLAG antibody. Cell lysates and immunoprecipitates were immunoblotted with the indicated antibodies.