Fig. 4: The deubiquitinase OTUD1 is involved in TAM-mediated stabilization of FGL1.

A Mass spectrometry (MS) analysis to explore different FGL1-binding proteins in 293T cells cocultured with TAMs. The representative candidates are listed. OTU deubiquitinase 1 (OTUD1) was identified. B Co-IP analysis of the interaction between exogenously overexpressed HA-OTUD1 and Flag-FGL1 in 293T and MEF cells. C GST pull-down assay analysis of the interaction between His-OTUD1 and GST-FGL1 proteins in vitro. D Co-IP analysis of the interaction between HA-Ub and Flag-FGL1 in OTUD1 knockdown or control MC38 and HT29 cells pretreated with Baf-A1 (100 nM, 24 h). E IB detection of FGL1 and OTUD1 expression in indicated cells. F Co-IP analysis of the interaction between HA-Ub and Flag-FGL1 in Myc-OTUD1-overexpressing or control MC38 and HT29 cells pretreated with Baf-A1 (100 nM, 24 h). G IB detection of FGL1 and HA-OTUD1 expression in indicated cells. H Co-IP analysis of the interaction between HA-Ub and Flag-FGL1 in indicated cells pretreated with Baf-A1 (100 nM, 12 h), and then incubated with TAM-CM for 16 h. I IB detection of FGL1 and OTUD1 expression in indicated cells incubated with TAM-CM. J IB detection and quantification of FGL1 expression in the indicated cells incubated with TAM-CM for 16 h following treatment with CHX for different times, n = 3 biologically independent experiments. K Co-IP analysis of the interaction between HA-Ub and Myc-FGL1 in 293T cells overexpressing Flag-OTUD1, Flag-OTUD1 (C320S) or control (EV). L Schematic diagrams of OTUD1 displaying the positions of different domains. M Co-IP analysis of the interaction of different truncations of Flag-OTUD1 with Myc-FGL1 in 293T cells. N Representative images and quantification of the indicated cells cocultured with activated OT-1 T cells for 8 h and then subjected to crystal violet staining, n = 3 biologically independent experiments. The ratio of MC38-OVA cells to OT-1 T cells was 5:1. IB experiments in (B–K, M) were repeated three times, the data are representative of three biologically independent experiments. The data in J, N are presented as the mean ± SD. The P value was determined by one-way ANOVA (N) and two-tailed unpaired Student’s t test (J).