Figure 7.

PHA-767491 modulates TCR signaling. (A) PHA-767491 treatment suppresses Erk phosphorylation. Immunoblots of OT-I CTL that were not-treated (–) or were treated (+) with PHA-767491 were stimulated with K^b-OVA tetramers for the indicated times. Normalized values of individual bands are indicated below the respective bands. Representative blots of at least three independent experiments are shown. (B) The ratio of the band intensities of the phosphorylated proteins to the respective total proteins from (A) are shown in the bar charts, depicted as mean ± SEM. Values represent fold change, normalized to the DMSO-treated sample values for the respective time points. (C) PHA-767491 does not impair Ca²⁺ flux. Peripheral lymphocytes were either pre-treated with PHA-767491 or not and were incubated with biotinylated anti-CD3 antibodies. Lymphocytes were first cross-linked using streptavidin, followed by an exogenous addition of CaCl₂ solution, and finally treated with ionomycin. (D) PHA-767491 affects TCR recycling in stimulation assays. Peripheral lymphocytes and thymocytes were stimulated with plate-bound anti-CD3 antibody or anti-CD3/CD28 beads, respectively, for 3 or 17 h. Values indicated in the histograms represent the MFI. (E) Cdc7 association with Dbf4 and MCM2 is reduced in TCR stimulated T cells treated with PHA-767491. Serum-starved Jurkat cells that were not-treated (–) or treated (+) with PHA-767491 were seeded into wells coated with plate-bound anti-CD3 antibodies and incubated for the indicated times and lysed. Anti-Dbf4 antibodies were used to immunoprecipitate Dbf4 from the cell lysates. Samples were immunoblotted for immunoprecipitated Dbf4 and co-immunoprecipitated Cdc7 and MCM2. Data shown is representative of at least three independent experiments. Bar charts are represented as mean ± SEM. Statistical significance was determined by unpaired two-sided Student's t-test (n.s, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001).