Recombinant protein

Naïve CD4⁺ T cells were sorted from the spleens and lymph nodes with the MACS CD4⁺CD62L⁺ T Cell Isolation (30–093-227) Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured under neutral (TH0) conditions in supplemented IMDM medium in the presence of activating antibodies (5 μg/ml plate-coated anti-CD3 and 1 μg/ml soluble anti-CD28) and iTreg polarizing cytokines: TGF-β [5 ng/ml], human IL-2 [20 ng/ml], αIL-4 [2 μg/ml], αIFN-γ [2 μg/ml] and αIL-12 [2 μg/ml].
Recombinant proteins (recombinant human IL-2 and TGF-β) and blocking antibodies (anti-mouse IL-4, anti-mouse IFN-γ, anti-mouse IL-12) for in vitro cell differentiation were purchased from eBioscience (San Diego, California,USA).

In vitro kinase assays were performed as previously described by us^{82 (link)}. Briefly, 10 ng of recombinant protein (ThermoFisher Scientific) was diluted in kinase buffer (20 mM HEPES [pH 7.5], 10 mM MgCl₂, 1 mM EGTA, 0.01% Brij35, 0.02 mg/ml BSA, 0.1 mM Na₃VO₄, 2 mM DTT, 1% DMSO and incubated with the indicated concentrations of 108600 at room temperature for 30 min. Kinase reactions were initiated by the addition of 1 μg substrate and mixture of 10 µM ATP (Sigma–Aldrich) and 100 µCi ³³P-γ-ATP (PerkinElmer). The reactions were incubated for 2 h at 25 °C and subsequently spotted onto P81 ion exchange filter paper. The filters were then thoroughly washed in 0.75% phosphoric acid to remove unbound phosphate and the level of substrate radioactivity quantitated using a scintillation counter. Values were plotted as a function of log drug concentration and IC₅₀ values determined by plotting sigmoidal non-linear regression curves with a variable slope. Peptide substrates used were as follows: CK2α and CK2α2: RRRDDDSDDD; TNIK: RLGDKYKTLRQIRQ; DYRK1: RRRFRPASPLRGPPK.

For Western blot analysis, 100 µg of secretome or 10 ng of recombinant protein (marca commercial) were digested with 20 µg/mL of Proteinase K (ThermoFisher) in a final volume of 20 µL. Samples were incubated at 37 °C with agitation O/N. Then, 1 µL of PMSF 100 nM was added to inactivate Proteinase K. For LC-MS/MS proteomic analysis, 100 µg of secretome was digested following the same protocol. Samples were filtered with 10 kDa amicon (Millipore) to remove the digested peptides since we were interested in analyzing the proteolytic-resistant proteins.

B cells were in vitro activated using LPS (10 µg/ml), an agonistic mAb against CD40 (αCD40) (10 µg/ml; clone FGK45), a combination of these two (10 µg/ml LPS and 10 µg/ml αCD40), IL-21 (100 ng/ml recombinant protein; eBiosciences) or by α-CD40 followed by LPS (10 µg/ml) during the final 5 hours of an 48 hours culture as described in literature [9] (link), [12] (link), [16] (link). The cells were washed extensively before being used in other assays. All cultures took place in IMDM supplemented with 5×10⁻⁵ M 2-mercaptoethanol, penicillin (500 units/ml) and streptomycin (50 µg/ml; all from Gibco) at 37°C and 5% CO₂ in either 96-well plates (1×10⁵/well) or 24-well plates (2×10⁶/well).