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. 2020 Apr 2;27(5):571–585.e6. doi: 10.1016/j.chembiol.2020.03.004

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IFITM3 Ubiquitination Is Important for Interaction with VCP

(A) CoIP of HA-IFITM3 with FLAG-VCP. HEK293T cells were transfected with HA-IFITM3 and/or FLAG-VCP, and cell lysates were immunoprecipitated with anti-FLAG resin for western blot analysis. The red asterisks indicate IFITM3 ubiquitination bands with high molecular weights.

(B) CoIP of endogenous IFITM3 with FLAG-VCP. HeLa cells were transfected with FLAG-VCP, and cell lysates were immunoprecipitated with anti-FLAG resin for western blot analysis.

(C) Analysis of ubiquitination status of IFITM3 and lysine mutants. HEK293T cells were transfected with HA-IFITM3, single lysine mutants (i.e., K24A, K83A, K88A, and K104A), or the quadruple lysine mutant K∗A, lysed for anti-HA IP, and analyzed by western blot.

(D) CoIP analysis of HA-IFITM3 and lysine mutants with endogenous VCP and its cofactors. The anti-HA immunoprecipitates were prepared as in (C) and analyzed by western blot.