Silvestrol reduces eIF4A cap-binding activity, protein translation, and oncoprotein expression in activated human splenic B cells. (A) Protein lysates from human splenic B cells were harvested 24 hours after treatment with Isotype + Vehicle, Isotype + 10 nM Silvestrol, anti-BCR + Vehicle, or anti-BCR + 10 nM Silvestrol and incubated with m7GTP Sepharose beads overnight at 4°C. Western blot analysis was used to observe eIF4A and eIF4E binding to m7GTP. Data are representative of 3e independent experiments. (B) Twenty-four hours after treatment, cells were incubated with 1 mCi L-[³⁵S]methionine and l-[³⁵S]cysteine, and lysates were harvested and analyzed for radiolabel incorporation. Densitometry was used to quantify radiolabel incorporation, and data are means ± SEM of 3 different experiments. (C) Protein levels of CARD11, BCL10, MALT1, c-Myc, and CCND2 were measured by western blotting. GAPDH served as a loading control. (D) Densitometry was used to quantify protein expression, and data are means ± SEM of 3 independent experiments (3 individual spleens). (E) Cells were fractionated through sucrose gradients, and the relative distribution of CARD11 (normalized to GAPDH) was analyzed by quantitative reverse transcription-polymerase chain reaction analysis of RNA in each of the 10 gradient fractions. Data are representative of 2 independent experiments (2 individual spleens; *P < .05).