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. 2022 Jun 8;221(7):e202108015. doi: 10.1083/jcb.202108015

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© 2022 Wang et al.

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Figure S1. — ORF10 specifically stimulates CUL2^ZYG11B activity. (A) ZYG11B could interact with ORF10. Purified ORF10-FLAG was used to pull down GST-ZYG11B. (B) ELOC could not bind with ORF10. Purified ORF10-FLAG was used to pull down 6 × His-ELOC-HA. (C) CUL2 could not interact with ORF10. Purified ORF10-FLAG was used to pull down CUL2-HA. (D) RBX1 could not bind with ORF10. Purified ORF10-FLAG was used to pull down 6 × His-RBX1-HA. (E) Reconstitution of the CUL2^ZYG11B ubiquitination system. Either E1, E2, ZYG11B-CUL2 immunocomplex, or Ub were withheld from the CUL2^ZYG11B ubiquitination system in vitro. The reaction products were detected by immunoblotting with anti-ubiquitin and FLAG antibodies. (F) ORF10 stimulates the ubiquitination activity of CUL2^ZYG11B. Either ORF10 or BSA was added to the CUL2^ZYG11B ubiquitination system in vitro, which contains E1, E2, ubiquitin, ATP and ZYG11B-CUL2 immunocomplex. The reaction products were detected by immunoblotting with anti-ubiquitin and FLAG antibodies. (G) ORF10 is essential for the stimulation of CUL2^ZYG11B ubiquitination activity. Either E1, E2, ZYG11B-CUL2 immunocomplex, Ub or ORF10 were withheld from the CUL2^ZYG11B ubiquitination system in vitro. The reaction products were detected by immunoblotting with anti-ubiquitin antibody. (H) VHL could not bind with ORF10. Purified ORF10-FLAG was used to pull down 6 × His-VHL-MYC. (I) ORF10 could not stimulate the ubiquitination activity of CUL2^VHL. Either ORF10 or BSA was added to the CUL2^VHL ubiquitination system in vitro, which contains E1, E2, ubiquitin, ATP, and CUL2^VHL. The reaction products were detected by immunoblotting with anti-ubiquitin antibody. (J) ORF10 has no effect on the ubiquitination activity of CUL2^VHL. Either ORF10 or BSA was added to the CUL2^VHL ubiquitination system in vitro, which contains E1, E2, ubiquitin, ATP, and CUL2^VHL. The reaction products were collected at the indicated times, and reaction products were detected by immunoblotting with anti-ubiquitin and FLAG antibodies. Source data are available for this figure: SourceData FS1.