The correlation of α4 expression and rapamycin sensitivity. (A) Expression of α4 in transfectants was shown by Western blot analysis. Jurkat transfectants expressing mα4 or neomycin-resistant gene, parental Jurkat, and mouse B-cell line WEHI 231 were lysed in lysis buffer containing 1% NP-40. The immunoblot was developed with anti-mα4 Ab (left side) or anti-hα4 Ab (right side). Anti-hα4 Ab recognizes both hα4 and mα4 proteins. The migrations of mouse and hα4 are as indicated. (B) Cells were cultured at 1 × 104/well in 200 μL of medium with various concentrations of rapamycin for 48 hours. Relative cell numbers were analyzed by WTS-1 assay. Results are shown as the percentage of the control culture without rapamycin. Data are representative of 4 independent experiments and are shown as the mean of duplicate samples ± standard deviations. (C) Jurkat cells were cultured at 1 × 105/mL with various concentrations of serum as 10%, 2%, or 0.4% for 72 hours. Rapamycin sensitivity was measured by the WTS-1 assay as described above. (D) Jurkat cells were cultured in the medium that contained either 10%, 2%, or 0.4% of FCS for 72 hours. The amounts of PP2Ac bound to α4 were detected after immunoprecipitation with anti-hα4 Ab, followed by anti-PP2Ac Western blot analysis. Total amounts of PP2Ac were detected using whole cell lysate (WCL). Columns below the bands show the arbitrary units to indicate the relative intensity of the bands as determined by a densitometer.
