Fig. 2: STUB1 destabilizes cell-surface IFNγ-R1 in JAK1-dependent and JAK1-independent manners.

a Proteomic profiling of D10 cells expressing sgCtrl or sgSTUB1. Highlighted proteins are differentially regulated in two cell lines (Supplementary Fig. 2e). b Immunoblot of D10 (left) and SK-MEL-147 (right) cells lines expressing sgCtrl or sgSTUB1. Whole cell lysates (WCL) were immunoblotted for indicated proteins (TUB is tubulin). Representative of three biological replicates. c Immunoblot of D10 cells ectopically expressing indicated constructs. WCL were immunoblotted for indicated proteins (TUB is tubulin). Representative of three biological replicates. d Flow cytometric quantification of IFNγ-R1 expression in D10 cells ectopically expressing indicated constructs. e qPCR analysis for IFNGR1 expression in D10 cells ectopically expressing indicated constructs. IFNGR1 expression was normalized to EGFP-expressing cells using the ΔΔCT method. f Immunoblot of parental D10 cells, D10 IFNGR1-KO clones and JAK1-KO clones expressing sgCtrl or sgSTUB1. WCL were blotted for indicated proteins (TUB is tubulin). Representative of three biological replicates. g Densitometric quantification of IFNγ-R1 protein levels (relative to loading control and normalized to D10 parental sgCtrl-expressing cells) from f. h Schematic depiction of STUB1 and its functional domains. i sgSTUB1-expressing HEK293T cells were transfected with Myc-tagged JAK1, V5-tagged IFNγ-R1, and the indicated FLAG-tagged STUB1 variants. Left: Immunoblot of the WCL of the indicated samples. Right: Immunoblot of immunoprecipitation (IP) samples. WCL and IP samples were blotted for indicated proteins (TUB is tubulin). ISO Isotype control. j Flow cytometric quantification of IFNγ-R1 expression in sgCtrl-expressing D10 cells ectopically expressing EGFP (control) and sgSTUB1-expressing D10 cells ectopically expressing either EGFP, wildtype STUB1 (WT), or the indicated STUB1 variants. All ectopically expressed proteins were FLAG-tagged. k Immunoblot of sgCtrl-expressing D10 cells ectopically expressing EGFP and sgSTUB1-expressing D10 cells ectopically expressing either EGFP, wildtype STUB1, or the indicated STUB1 variants. WCL were blotted for indicated proteins (TUB is tubulin). Representative of three biological replicates. Mean±SD in d, g: ordinary one-way ANOVA for three biological replicates with Tukey post hoc testing. ****p < 0.0001 (d), ***p = 0.0004, ****p < 0.0001 (g). Mean ± SD in (e): n.s. p = 0.8001, ****p < 0.0001, ordinary one-way ANOVA for three biological replicates with Dunnett’s post hoc testing. Mean±SD in j: ****p < 0.0001, ordinary one-way ANOVA for three biological replicates with Sidak’s post hoc testing.