, HA-Nck WT and HA-Nck R311K mutant constructs were
, Amaxa Biosystems), were electroporated with 1 ?g of the indicated expression vector using the V-024 programme of the Nucleofector. For luciferase assays, 10 7 cells were electroporated (at 260 V, 960 µF) in 0.5 ml RPMI 1640 supplemented with 20% FCS with 2 ?g NF-?B luciferase or 10 ?g NF-AT luciferase together with 5 ?g pEGFP and each indicated expression vector, keeping the total amount of DNA constant (40 µg) with empty vector. 24 h after transfection, cells were stimulated with Dap3 or Dap3/B7 or 5-3.1 or 5-3.1/B7 cells pre-pulsed with SEB (1 ?g ml ?1 ) at 37°C for 6 h. Luciferase activity was measured according to the manufacturer's instruction (#E1500, Promega)
, 1 mM EGTA, 1 mM MgCl 2 , 50 mM NaF, 10 mM Na 4 P 2 O 7 ) in the presence of inhibitors of proteases and phosphatases (10 ?g ml ?1 leupeptin, 10 ?g ml ?1 aprotinin, 1 mM NaVO 4 , 1 mM pefablock-SC), Extracts were precleared for 1 h with Protein-A (Amersham) or Protein G Sepharose beads, vol.20
, Proteins were resolved by SDS-PAGE and blotted onto nitrocellulose membranes. Blots were incubated with the indicated primary antibodies, extensively washed and after incubation with horseradish peroxidase (HRP)-labelled goat anti-rabbit (#NA934V, 1:5000 dilution) or HRP-labelled goat anti-mouse (#NA931V, 1:5000 dilution) (Amersham), or (HRP)-labelled donkey anti-goat Abs, Abs (CD28.2, 2 ?g per immunoprecipitation) pre-adsorbed on Protein-G Sepharose beads (20 ?l per immunoprecipitation) or rabbit anti-hamster Abs
, × 10 5 cells per well) either unstimulated or stimulated with crosslinked anti-CD28.2 Ab (2 ?g ml ?1 ) or adherent Dap3/B7 cells fixed with paraformaldehyde to avoid detachment and secretion. A cytometric bead-based immunoassay (#558277, CBA FLEX SET, BD Biosciences) was used to measure IL-8. Data were analysed with the FCAP Array, software (Soft Flow Hungary Ltd
, TaqMan Universal PCR Master Mix, human: IL-8, IL-2, IL-6, IFN-?, TNF, IL-1? and endogenous GAPDH, mouse: IL-2, IL-6, IFN-?, TNF and endogenous RLP32 primer/probe sets were purchased from Applied Biosystems. The relative quantification was performed using the comparative C T method. The median value expressing hCD28 WT, or mCD28 WT, or CD28P 212 A mutant, or chimera CD28 WT, or chimera CD28 A 210 P mutant were transfected for 24 h with 20 ?g of GFP or cherry constructs, seeded on cover glasses for 15 min at 37°C, fixed by 2% paraformaldehyde and permeabilised by 0.1% saponin in PBS containing 1% BSA. Filamentous actin (F-Actin) was stained by using phalloidin-633 (#A22284, 1:75 dilution, Molecular Probes). Confocal observations were performed using a Leica DMIRE (Leica Microsystems, ) from 2 × 10 6 cells and RNeasy MicroKit, vol.74004
, × 10 6 ) were incubated for 5 min at 37°C with Dap3/B7 (1.2 × 10 6 ) or RAW-264.7 cells in a final volume of 70 ?l of RPMI, then diluted in 500 ?l RPMI and analysed by FACS. Conjugates were identified on a total of 10 5 GFPpositive events by gating for SSC and FSC and expressed as mean percentage ± SD of triplicate samples. An example of the gating strategy used is reported in Supplementary Fig. 13. Statistical analysis. The sample size was chosen based on previous studies to ensure adequate power. Parametrical statistical analysis (mean and SD) was performed to evaluate differences between continuous variables through Prism 5.0 (GraphPad Software, Conjugate formation was measured as previously described 6 . Briefly, CH7C17 Jurkat cells expressing hCD28 WT, or mCD28 WT or CD28 mutants were transfected with pEGFP construct and transfectants
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