Skip to main content
. 2022 Aug 18;12:14087. doi: 10.1038/s41598-022-18404-4

Figure 3.

Figure 3

Pharmacological inhibition of STUB1 with expressed biologic phenocopies the genetic knockout. (a, b) Validation of the binding of synthetic peptides to STUB1 (aa25–aa153) by isothermal titration calorimetry (a) and thermal shift assay (b). Representative of three independent experiments (a). Data are mean ± s.d. of six replicates derived from three independent experiments (b). (c) Competitive fluorescence polarization assay. Synthetic peptides were assessed for their ability to compete with 15 nM of tracer peptide (5-FAM-SSGPTIEEVD) for binding to 1 µM STUB1 (aa25–aa153). Data are mean ± s.d. of six replicates derived from two independent experiments. (d) Design of the inhibitory biologic by grafting the peptide (SIWWPD) to the C-terminus of an mCherry2 (red) scaffold. The fused peptide blocks the function of the tetratricopeptide repeat domain (blue) of STUB1 (PDB code 2C2L) and inhibits its substrate binding. U-box domain (orange) which recruits the E2 ubiquitin-conjugating enzyme is not affected. (e) Generation of tumour cell lines stably expressing the biologic or its control. Plasmid encoding the biologic was electroporated into tumour cells, followed by antibiotics selection of the stable clones. The mCherry2-positive cells (red dotted box) were further gated for mCherry2hi population (top 50th percentile, red box). Gating example represents IFNγ-treated B16-F10 stable cell lines. (f, g) Flow cytometry analysis of the relative cell surface level of IFNGR1 (f) and MHC-I (g) expressed by the mCherry2hi population in B16-F10, A375 or A549 cells. The cells were either untreated or treated with mouse IFNγ (0.03 ng ml−1) or human IFNγ (0.01 ng ml−1) for 24 h. The expression levels were normalized to the average value of the control (mCherry2-SIWWHR). n = 5 biological replicates from two independent experiments (f–g). Bars are mean with all data points (f–g). P values were determined by ordinary two-way ANOVA in each cell type with Sidak’s multiple comparisons test, ****P ≤ 0.0001 (f–g). (h) Co-immunoprecipitation (co-IP) of FLAG-mCherry2-peptide and STUB1 from the cellular lysate of B16-F10 using anti-FLAG antibody. Synthetic peptide (SIWWPD) was added into the co-IP mixture to assess specificity of the interaction. Blot is representative of three independent experiments.