Fig 2: C121-mediated multimerization governs the stability and localization of unglycosylated 4-1BB. (A) Jurkat cells were transduced with lentivirus encoding 4-1BB WT, C121A, 2NQ, and 2NQ/C121A, respectively. The membrane levels of 4-1BB in each group were compared by flow cytometry. The percentages of positive events were included in the plots. (B) The quantitative results of membrane 4-1BB level in each group in (A) (n = 3). (C) The immunoblotting analysis of total 4-1BB from each group of cells in (A). N.S., not significant; ***, p < 0.001 (Two-tailed student’s t-test); MFI, mean fluorescence intensity.

Fig 3: Regulation of CD137 and CKS2 protein expression in ECs by PTX and DEX.(A). Immunoblotting of CD137 in ECs. Lysates of ECs treated with vehicle, DEX, or PTX for 24 hours were probed for CD137. HSP90 was used as loading control. Representative images of three independent experiments are shown.(B). Immunoblotting of CD137 in ECs. Lysates of ECs treated with vehicle, 5 nM PTX, or 5 nM PTX + 1 μM or 10 μM DEX for 24 hours were probed for CD137. HSP90 was used as loading control. Representative images of three independent experiments are shown.(C). Normalized CD137 expression. Bars indicate mean of three independent experiments ± SEM. Student’s t test was performed between each treatment and vehicle control. *P = 0.042, **P = 0.004, ##P = 0.002, ###P < 0.001.(D). Normalized CD137 expression. Bars indicate mean of three independent experiments ± SEM. Student’s t test was performed between each treatment and vehicle control.***P = 0.001, ***P = 0.003, ####P < 0.001.(E). Immunoblotting of CKS2 in ECs. Lysates of ECs treated with vehicle, DEX, or PTX for 24 hours were probed for CKS2. HSP90 was used as loading control. Representative images of three independent experiments are shown.(F). Immunoblotting of CKS2 in ECs. Lysates of ECs treated with vehicle, 5 nM PTX, or 5 nM PTX + 1 μM or 10 μM DEX for 24 hours were probed for CKS2. HSP90 was used as loading control. Representative images of three independent experiments are shown.(G). Normalized CKS2 expression. Bars indicate mean of three independent experiments ± SEM. Student’s t test was performed between each treatment and vehicle control. *P = 0.036, ***P = 0.019, ###P < 0.001.(H). Normalized CKS2 expression. Bars indicate mean of three independent experiments ± SEM. Student’s t tests were performed between pairs of treatments. ***P = 0.036, ****P < 0.001, ####P < 0.001.

Fig 4: N-glycosylation plays an essential role in determining 4-1BB stability. (A) HEK293T cells were transfected with Flag-tagged 4-1BB WT and 2NQ followed by CHX-chase assay to compare their stability. (B) The quantification of the remaining 4-1BB at each time point (n = 3). The percentage values of band intensity were normalized to time 0 h. 50% degradation is indicated by the dashed line. (C) Immunoblotting analysis of the MG-132 effect on 4-1BB WT and 2NQ. HEK293T/4-1BB WT and HEK293T/4-1BB 2NQ cells were treated by 10 μM MG-132 for 0, 4, and 8 h followed by immunoblotting assay. (D) Comparison of polyubiquitination level between WT and 2NQ 4-1BB. Flag-tagged 4-1BB and HA-tagged Ub were co-transfected to HEK293T cells followed by in vivo ubiquitination assay 48 h after transfection. IP, immunoprecipitates. **, p < 0.01; ***, p < 0.001 (Two-tailed student’s t-test).

Fig 5: 4-1BB is a heavily N-glycosylated protein with two modified sites. (A) Alignment of mammalian 4-1BB sequence around the N-glycosylation sites. The N-X-S/T motif is outlined by the red square. (B) Immunoblotting analysis of 4-1BB undergoing deglycosylation treatment by PNGase F. (C) MALDI-MS profiling of permethylated N-glycans released from PNGase F-treated recombinant 4-1BB protein. The masses of indicated glycan species represents the [M + Na+] values. SP, signal peptide; ECD, extracellular domain; TM, transmembrane domain; ICD, intracellular domain; CDR, cysteine-rich domain; EV, empty vector; 4-1BB (G), N-glycosylated 4-1BB; 4-1BB (NG), non-N-glycosylated 4-1BB.