Makorins interact with and are stabilised by USP9X. (A) HEK293T cells were transfected with FLAG-MKRN1, FLAG-MKRN1-H307E, MKRN2-FLAG, or FLAG alone (pCMV-Tag2B) and cell lysates were subjected to immunoprecipitation (IP) with FLAG-antibody coupled agarose beads. IPs were probed alongside 2% of the input as indicated. Flag-MKRN1 H307E bears an inactivating mutation. PABP, polyA binding protein; eS10, 40S ribosomal subunit. Results are representative of three independent experiments. Arrow indicates ZNF598; arrowheads indicate FLAG-Makorins (MKRN). (B) HCT116 or HCT116 USP9X^−/0 cells were treated for the indicated times with 100 µg/ml cycloheximide (Chx). (C) Steady-state levels of MKRN1 in HCT116 compared with HCT116 USP9X^−/0 cells. Error bars indicate the standard deviation of four independent experiments, where the higher molecular weight isoform of MKRN1 has been quantified. (D) Quantitative RT-PCR reactions for MKRN1 (normalized to actin) were performed with cDNA derived from the indicated cell lines. The mean of three independent biological replicates is shown, and error bars indicate the standard deviation. (E) (K^AAA)₂₁ WT cells were treated for the indicated times with 100 µg/ml cycloheximide with or without FT709. (F) FT709 (10 µM) responsive markers in other cell types: A549 lung adenocarcinoma cells and U2OS osteosarcoma cells. TOMM20 here serves an alternative loading control for the upper set of panels. (B, E, and F) Arrows indicate two isoforms of MKRN1. *, a nonspecific band. Two-tailed Student’s t test; ***, P < 0.001. Untrf., untransfected.