Fig. 5: The CUL5 E3 complex targets TCR and IL2 signaling in CD8⁺ T cells.

a DIA-MS signals of enriched proteins in TCR-activated mouse primary CD8⁺ T cells transduced with CUL5-HA overexpressing vector (CUL5-HA) vs empty control vector (NC) identified by anti-HA co-IP MS (Cut-off: p value < 0.05 and fold change >1.5 in multiple t-test). b Western blot analysis of PCMTD2 expression in primary mouse CD8⁺ T cells with NC or PCMTD2 KO. HSP90 as an internal control. This analysis was repeated three times. c, Flow cytometry analysis of GZMB (as positive % in cytokine expansion condition, Left) and IFNg (as MFI in 6-hour anti-CD3/CD28 stimulation condition, Right) expression of NC (Black) or PCMTD2 KO (Red) primary mouse CD8⁺ T cells. Data are shown as mean + SEM (Two-sided unpaired t-test, n = 3). d Heatmap of signaling pathway enrichment of CUL5 KO CD8⁺ T cells compared to NC cells based on differentially expressed proteins using DIA-MS quantification. e DIA-MS signals of proteins identified by the total protein MS analysis of mouse primary CUL5 KO (Red) and NC (Black) CD8⁺ T cells post TCR stimulation. Data are shown as mean ± SEM (Two-sided unpaired t-test, n = 3). f Flow cytometry analysis of p-ERK1/2 expression in NC or CUL5 KO primary mouse CD8⁺ T cells after anti-CD3 plus anti-CD28 stimulation. Data are presented (Left) as mean ± SEM (Two-sided unpaired t-test, n = 3). g Flow cytometry analysis of p-STAT5 expression in NC or CUL5 KO primary mouse CD8⁺ T cells after 16-hour anti-CD3 plus anti-CD28 stimulation supplied with 5 ng/ml hIL2. Data are presented (Left) as mean ± SEM (Two-sided unpaired t-test, n = 3). h, i, Flow cytometry analysis of GZMB and IFNg in NC (Black and Blue) or CUL5 KO (Red and Purple) primary CD8⁺ T cells in 16-hour h, cytokine culture (IL2/IL7/IL15) or i, anti-CD3 plus anti-CD28 stimulation with (Blue and Purple) or without (Black and Red) 250 nM TAS4464. Data are shown as mean ± SEM (ns, not significant; two-sided unpaired t-test, n = 3). Datum points represent biological replicates.