Figure 4

Domain requirements for Sam68ΔC inhibition of gp120 protein synthesis from pgTat. a) Illustration of Sam68ΔC domain structure, and various mutants thereof. Vertical black bars represent the proline rich domains; hatched boxes represent the RGG boxes; the horizontal bars represent the GSG domain; and the speckled box represents the KH domain. At the bottom is the illustration of the unspliced pgTat reporter construct, which encodes gp120. The black bar below pgTat indicates the position of the RPA probe used and the protection products expected. b) 293T cells were transfected with pgTat, Rev and the indicated Sam68ΔC mutants. Forty-eight hours post-transfection, cells were harvested and RNA and protein extracted for analysis. A representative western blot indicating Env (gp120) expression in the presence of Sam68ΔC and various mutants thereof is shown. Blots were reprobed with anti-myc to confirm protein expression and anti-tubulin to normalize for loading. c) RNA analysis of pgTat by RNase protection assay. The four protection products are indicated: unspliced-uncleaved (us-uc), unspliced-cleaved (us-c), spliced-uncleaved (s-uc) and spliced-cleaved (s-c). gp120 is translated from the us-c isoform of the RNA. 20 μg of total RNA was input to the assay.