Fig. 1.

CD28 and TCR uniquely regulated NF-κB. (a) 1G4 CD28 deficient Jurkat cells reconstituted with either empty plasmid (vector) or WT (CD28) untagged plasmid were used to measure NF-κB IL-2 promoter luciferase (firefly) activity in response to indicated ligating antibodies. Activity was measured by normalizing firefly luciferase counts to the background Renilla luciferase values, done in triplicates for each point as described in Materials and Methods section. All values are in relative luciferase units. (b) NF-κB activation in CD3⁺ human peripheral blood lymphocytes in response to engagement with indicated concentrations of CD3 (OKT3 clone) or CD28 (CD28.2 clone) alone or in combination. (c) Electromobility shift assay from nuclear fractions of C57BL/6 naïve T-cells as described in section 2. Briefly, a biotinylated NF-κB probe was used to detect DNA binding to nuclear fraction from unstimulated (lane 1) or anti-CD28 stimulated cells (lane 2). For specificity, anti-CD28 stimulated nuclear fraction was probed with unlabeled/cold NF-κB oligo (lane 3). Lane 4 is positive control (provided with kit). (d) NF-kB activation in primary T-cells isolated from WT or CD28 KO mice treated with control (RαH), anti-CD3 or anti-CD28 alone stimulating antibodies or in combination. Data in each sample is average of triplicates. All antibodies concentrations are in micrograms per milliliter of solution.