TcPUF6 is present in the ribosome-free fractions of sucrose
gradients. a, the positions of the 40 and 60 S subunits, the 80 S
ribosome monomer, and polysomes are indicated in the sucrose density gradient.
b–d, Western blots of the collected fractions (30
μl) probed with anti-TcPUF6 (1/500) (b), anti-TcTif34 (1/500)
(c), and anti-PEPCK (1/250) sera (d). Transfected parasites
overexpress a TcPUF6-tagged protein. e, Western blot analysis of
protein extracts from epimastigotes (lane 1), epimastigotes
transfected with the pTEX-TAPTAG-transfected clones (lane 2), and the
PUF6 A- and (4) B-transfected
clones (lane 3), with antiserum against TcPUF6 protein (1/500
dilution). f, protein extracts were also tested with an anti-CBP
serum (1/1000) (Upstate). g, the same blot was re-probed with an
anti-PEPCK serum (1/500). h, immunolocalization of T. cruzi
TcPUF6 in TcPUF6-TAPTAG-transfected epimastigote forms with anti-CBP antiserum
(1/100). Bars, 5 μm.