PBM is essential for IL-2-independent growth of CTLL-2 cells induced by Tax1. (A) CTLL-2 is a mouse T-cell line. This cell line was cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (RPMI-FBS), antibiotics and 0.5 nM recombinant human IL-2. To establish CTLL-2 cell lines expressing Tax or Tax mutant proteins, CTLL-2 cells (1 × 107) were suspended in 400 μl Opti MEM1 (Gibco BRL, Gaithersburg, MD), mixed with 20 μg of the vector plasmid (pβAIP) or with expression plasmids encoding Tax1 or Tax mutants, and then pulsed at 200 V and 975 F. The cells were seeded in 96 well plates 24 h after electroporation and cultured in RPMI-FBS containing 0.5 nM IL-2 and 2 μg/ml puromycin for 4 to 6 weeks. Puromycin-resistant cells were screened for the expression of Tax protein by Western blot analysis using mouse anti-Tax1 monoclonal antibody (TAXY-7)  as described previously . (B) CTLL-2/Vector, and two of each CTLL-2 clone expressing Tax1 or Tax mutant proteins were washed twice with phosphate-buffered saline (PBS) and cultured in IL-2-free medium for 3–5 days. Cell growth was measured by the trypan blue staining method using light microscopy.