Ki67 ki 67

The human 20S immunoproteasome core sample was purchased from Boston Biochem. The human i-20s at a concentration of 1.5 mg per ml was incubated with PKS21004 dissolved in DMF with a molar ratio of 1:250 ratio for 1 h at 37 °C, then the mixture was diluted in 50 mM HEPES, pH 7.6, 100 mM NaCl and 1 mM dithiotreitol to a final concentration of 0.1 mg per mL for cryo-EM study. Antibodies used for flow cytometry are described as below (clone, company): CD3 (UCHT1, Biolegend), CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD11c (3.9, Biolegend), CD14 (RMO52, Beckman Coulter), CD16 (3G8, Biolegend), CD19 (HIB19, BD), CD38 (LS198–4–3, Beckman Coulter), CD27 (O323, eBioscience), IgD (IA6–2, eBioscience), HLA-DR (L243, Biolegend), Ki67 (Ki-67, Biolegend).

Cell surface markers were stained for 30 min in the dark at 4 °C. Intracellular cytokine staining was performed using the ebioscience Foxp3 staining kit (ThermoFisher Scientific, Waltham MA) per manufacturer’s protocol. The following mouse antibodies from BioLegend (San Diego CA) were used: CD4 (GK1.5); CD8 (53-6.7); Tbet (4B10); Gata3 (16E10A23); Foxp3 (MF-14); IFNγ (XMG1.2); IL-10 (JES5-16E3); IL17 (TC11-18H10.1); and TGFβ (TW7-16B4). RORγt (AFKJS-9) was ordered from eBioscience (ThermoFisher Scientific). To show T cell reactivity, splenocytes were isolated from tumor bearing mice and cultured with 4T1 cells at a ratio of 5:1 (splenocytes to tumor cells) in the presence anti-CD107A/CD107B (ThermoFisher Scientific) and Monensin for 4 h. After 4 h, cells were stained for surface and intracellular markers described above. Flow cytometry analysis of T cell markers on human PBMCs was performed using the following clones: CD8 (RPA-T8); CD4 (SK3); Tbet (4B10); Ki67 (Ki67) from BioLegend; RORγt and granzyme B (GB11) from ThermoFisher Scientific.