Rapamycin-sensitive dissociation of PP2Ac and α4. (A) Rapamycin sensitivity was measured on the proliferation of Jurkat and Raji. Jurkat is sensitive to rapamycin treatment, but Raji is relatively insensitive. Relative cell proliferation was compared after measuring by WST-1 assay.31 (B) Rapamycin induces the dose-dependent dissociation of PP2Ac/α4 complex in rapamycin-sensitive Jurkat but not in rapamycin-resistant Raji. Cell lysates were prepared after the culture and the PP2Ac coprecipitated with α4 is detected by Western blot analysis. After detecting signals, the probe on the filter was stripped off according to the company's protocol (Amersham). The filter was then reprobed with anti-hα4 Ab to see the amount of hα4 protein in each lane. To further confirm the existence of similar amounts of PP2Ac and hα4 proteins, Western blot analysis was performed with the whole cell lysates (WCL). (C) Both Jurkat and Raji cells were treated with rapamycin (330 nmol/L) and the PP2Ac associated with α4 was detected by Western blot analysis. Cells were harvested at the time points indicated and the immunoblot was developed with anti-PP2Ac Ab. The migration of PP2Ac is as indicated. (D) Effect of FK506 was examined during the treatment of rapamycin. Varying concentrations of FK506 were added in the culture of Jurkat with rapamycin (10 nmol/L). The cells were harvested after 48 hours and lysed as described above. The α4-associated PP2Ac was detected on Western blot analysis. The PP2Ac signals were measured by the densitometric analysis and are shown as the relative intensities (%) of control culture in the absence of rapamycin. The amounts of hα4 immunoprecipitated with anti-hα4 Ab were controlled by reprobing the same filter (data not shown).