Cd38 ls198 4 3

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).

Polyclonal B–cell activation was carried out by stimulating 2 × 10⁶ PBMC/well in a total volume of 2 mL/well in 24–well plates with an activation cocktail consisting of 2.5 μg/mL Toll–like receptor 7/8 agonist (resiquimod [R848]; Sigma–Aldrich, St. Louis, MO) and 1000 IU/mL IL–2 (Proleukin, Novartis, the Netherlands).^{15 (link)} Peripheral blood mononuclear cell cultures were carried out in Iscove’s modified Dulbecca’s medium (Gibco Invitrogen, Paisley, UK) containing 10% fetal bovine serum (Gibco Invitrogen) and 100 U/mL penicillin with 100 μg/ml streptomycin (Gibco Invitrogen). Supernatants harvested at day 6 (d6) or day 10 (d10) were kept at −20°C until further use. Flow cytometry was performed before (d0) and after (d6, d10, and d14) polyclonal activation according to standard protocols using the following antibodies (clone): CD19 (A07770), CD27 (1A4CD27), CD38 (LS198–4–3) and CD45 (J33) (all from Beckman Coulter, Woerden, the Netherlands).