FIG. 7.

Expression of CRAM or truncated CRAM in different bloodstream-form-transformed cell lines. (A) Northern blot analysis. Total RNAs from wild-type procyclic trypanosomes (P), wild-type bloodstream-form trypanosomes (BS), and BS-CRAMH23H (H), BS-CRAMH23HB (HB), BS-CRAM-40 (-40), BS-CRAM-29 (-29), BS-CRAM-19 (-19), and BS-CRAM-4 (-4) cell lines were separated in 1% formaldehyde agarose gels. The blot was hybridized to a 5′ CRAM probe (Fig. 1). The final posthybridization wash was performed in 0.1× SSC–0.1% SDS at 65°C. The bottom panels represent hybridization with the β-tubulin gene probe indicating the relative amount of RNA loaded in each lane. (B) Western blot analysis. Total protein lysates (∼2 × 10⁷ trypanosomes), derived from the wild-type procyclic trypanosomes (P), the wild-type bloodstream-form trypanosomes (BS), and BS-CRAMH23H (H), BS-CRAMH23HB (HB), BS-CRAM-4 (-4), BS-CRAM-19 (-19), BS-CRAM-29 (-29), and BS-CRAM-40 (-40) cell lines were size separated in 6% polyacrylamide gels and electrophoretically transferred to nitrocellulose filters. The blots were probed with anti-CRAM antibody (24). The same blots were later probed with anti-Tb-29 (right) (26) or the anti-procyclic antibody identifying two proteins of ∼43 to 45 kDa (left) (Lee, unpublished) to demonstrate equal loading of protein.