Regulation of IGF-1 by peripheral B cells in aging is mediated by TNF-α through IGFBP-1. (A) Sera collected from the indicated mice were analyzed for TNF-α by ELISA. Shown are results for individual mice and group means (n = 6-7 for each group). (B) Sera were collected from old hCD20Tg mice before and 14 days after B-cell depletion and analyzed for levels of TNF-α by ELISA. Shown are longitudinal results for individual mice (n = 3). (C) Purified splenic B cells from the indicated mice were analyzed for relative expression of TNF-α messenger RNA (mRNA) by quantitative polymerase chain reaction (qPCR) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Graph depicts results for individual mice (n = 9-11) and group mean ± SE. (D) Purified splenic B cells from the indicated mice were cultured in vitro for 24 hours. Supernatants were collected and analyzed for TNF-a by ELISA. Shown are results for individual mice and group means (n = 8-13 for each group). (E) Sera collected from the indicated mice were analyzed for IGFBP-1 by ELISA. Shown are results for individual mice and group means (n = 8-9 for each group). (F) Measurements of IGF-1, TNF-α, and IGFBP-1 for individual mice from the indicated groups were plotted in a 3-dimensional chart. Each group is clustered with a color-matched covariance ellipsoid centered around the mean of each group.