eIF4A cap-binding activity and protein translation is higher in IgG-expressing and IgG-activated B cells. (A) Human splenic lymphocytes isolated from motor vehicle collision patients between the ages of 41 and 88 years were sorted for IgG high/IgM low or IgG low/IgM high CD19⁺ B-cell populations. (B) Each population was treated with no treatment, goat anti-human IgG Fcγ, or goat anti-human IgM Fc5μ. Isotype-specific activation was then induced 30 minutes after primary antibody incubation by treating all conditions with anti-goat IgG. Twenty-four hours after activation protein lysates were harvested and analyzed for p-ERK (Thr202/Tyr204), total ERK, p-PDK1 (Ser241), total PDK1, p-MTOR (Ser2448), total MTOR, p-p70s6K (Thr389), total p70s6K, p-RPS6 (Ser235/236), RPS6, p-PDCD4 (Ser67), and total PDCD4. β-Actin served as a loading control, and data are representative of 2 independent experiments. (C) Twenty-four hours after activation, protein lysates were incubated with m7GTP Sepharose beads overnight at 4°C, and western blot was used for analysis of eIF4E and eIF4A cap-binding activity. Densitometry was used to quantify protein expression, and data are means ± SEM of 3 independent experiments. (D) Twenty-four hours after isotype-specific activation, 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.