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. 2016 Jan 28;25(7):1392–1405. doi: 10.1093/hmg/ddw021

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Figure 3. — Itch catalyzes monoubiquitination of SMN. (A) FLAG-SMN, HA-Itch and HA-Itch (C830A) were expressed in 293T cells as indicated. SMN was immunoprecipitated with an anti-FLAG antibody under a denaturing condition, followed by immunoblotting of Ub and FLAG. Asterisks denote ubiquitinated SMN. (B) Similar to (A) except that Myc-Ub was expressed in certain combinations. (C) Reconstitution of Itch-mediated SMN ubiquitination using purified proteins. In addition to indicated proteins, all reactions contained ubiquitin, UbE1 and UbcH7. (D) Comparing Itch-mediated SMN ubiquitination in the presence of Ub or Ub(K0). All other conditions were similar to (C). Asterisk denotes oxidized SMN formed by disulfide bonds (40). (E) Ubiquitination reactions were similar to (D) except SMNΔ7 was used.