Usp9x and Usp24 associate with Mcl-1. (A) HCT116 Usp9x^+/+ (wild-type) and Usp9x^−/− cell lysates were subjected to immunoprecipitation (IP) with normal immunoglobulin G (IgG), anti-Mcl-1, anti-Usp9x, or anti-Usp24 before immunoblotting (IB) the pulldown for Mcl-1, Usp24, or Usp9x (top 2 panels). H929 cell lysates were immunoprecipitated with normal IgG, anti-Mcl-1, or anti-Usp24 and immunoblotted for Mcl-1 or Usp24 (bottom panel). (B) Organization of the Usp9x and Usp24 constructs used in pulldown experiments and summary of their Mcl-1 binding activity. The position of the catalytic domain ubiquitin C-terminal hydrolase (UCH) is shown by shading. Flag sequence and HA epitope in the constructs are positioned at the N-termini. Numbers and letters designate highlighted amino acids. (C) Flag-tagged control or Flag-tagged full-length and deletion constructs of Usp9x (illustrated in panel B) were expressed in HEK293T cells and subjected to Flag immunoprecipitation followed by immunoblotting of Mcl-1. (D) Flag-tagged control or deletion constructs of Flag-E1-Usp9x retaining the active site cysteine (C1566) or a catalytic domain mutant (CDM-C1566A) were expressed in HEK293T cells and subjected to Flag pulldown followed by Mcl-1 immunoblotting. (E) HEK293T cells were transfected with the constructs designated before the input lysates and anti-HA (Usp24) immunoprecipitates were subjected to immunoblotting with antibodies against Usp9x, Usp24, and Mcl-1. WB, western blotting.