Fig. 3. STUB1 drives proteasomal degradation of IFNγ receptor complex through IFNγ-R1^K285 and JAK1^K249 residues.

a Immunoblot of D10 cells expressing sgCtrl or sgSTUB1, treated with vehicle or 10 µM MG132 for 4 h. Whole cell lysates (WCL) were immunoblotted for indicated proteins (TUB is tubulin). Representative of three biological replicates. b Densitometric quantification of IFNγ-R1 protein levels (relative to loading control and normalized to vehicle-treated group) from a. c as in b for JAK1 protein. d Schematic depiction of reconstitution of IFNγ-R1^WT + JAK1^WT or IFNγ-R1^K285R + JAK1^K249R cDNAs in IFNGR1-KO + JAK1-KO D10 clones in either a sgCtrl- or sgSTUB1-expressing genetic background. e Immunoblot of IFNGR1-KO + JAK1-KO D10 clones, reconstituted with either IFNγ-R1^WT + JAK1^WT or IFNγ-R1^K285R + JAK1^K249R cDNAs, after four-hour treatment with vehicle or 10 µM MG132. WCL were immunoblotted for indicated proteins (TUB is tubulin). Representative of three biological replicates. f Densitometric quantification of IFNγ-R1 protein levels (relative to loading control and normalized to vehicle-treated group) from e. g as in f for JAK1 protein. h Immunoblot on WCL of IFNGR1-KO + JAK1-KO D10 clones reconstituted with constructs as outlined in d. WCL were immunoblotted for indicated proteins (TUB is tubulin). Representative of three biological replicates. i Fold change of IFNγ-R1 MFI (relative to IFNGR1-WT + JAK1-WT-expressing cells) in IFNGR1-KO + JAK1-KO D10 clones reconstituted with constructs as outlined in d. Bar chart represents an excerpt from Supplementary Fig. 3g. j Mass spectrometry-based quantification of STUB1-ubiquitinated JAK1 lysine residues after in vitro ubiquitination reaction of JAK1^233–332. Depicted lysine residues were also identified in ubiproteome profiling (Supplementary Fig. 3d). k Normalized abundance of ubiquitinated JAK1 lysine residues in sgCtrl and sgSTUB1-expressing D10 cells. l Model of STUB1-mediated proteasomal regulation of IFNγ-R1 and JAK1. Mean±SD in b, c, ordinary one-way ANOVA for three biological replicates with Tukey post hoc testing. **p = 0.0085 (b), ***p = 0.0007 (c). Mean ± SD in f, g, ordinary one-way ANOVA for three biological replicates with Sidak post hoc testing. *p = 0.0322 (f), **p = 0.0041 (g). Mean ± SD in i, *p = 0.036, ordinary one-way ANOVA for three biological replicates with Tukey post hoc testing. Mean±SD in j, k, ordinary one-way ANOVA for three experimental replicates with Sidak post hoc testing. *p = 0.0346 (j), ****p < 0.0001 (k).