Consequences of reducing SF3B2 expression. Consequences of reducing SF3B2 expression. (A) Normal quiescent human T cells were transfected with a pool of four siRNA duplexes against SF3B2 with non‐targeting siRNA as control. After stimulation with PMA/ionomycin for 72 h, the expression of SF3B2, phospho‐pRbs807/811, cyclin A2, cyclin D3, E2F‐1 and Cdc6 was detected by western blotting, with GAPDH as a loading control. (B) The expression of phospho‐mTORS2448, phospho‐4EBP1T37/46 and phospho‐eIF4Es209 was detected in T cells transfected and stimulated as in (A). (C) T cells were transfected with a pool of four siRNA duplexes against SF3B4 or with non‐targeting siRNA as control. The cells were stimulated with PMA/ionomycin and the expression of SF3B4, phospho‐pRbs807/811, cyclin A2, E2F‐1, Cdc6, phospho‐4EBP1T37/46 and phospho‐eIF4Es209 were detected by western blotting. (D) T cells transfected and stimulated as in (A, B) were fixed with ethanol, stained with PI (DNA content) and FITC (total protein content as a measure of cell size; see main text) and the proportion of cells in each of the cell‐cycle phases indicated in the first panel was determined by flow cytometry. A sample of cells transfected as in (A) was also sampled after 5 days in culture and analyzed in the same way. The percentages for this experiment are shown and the mean±s.e.m. for all repeat experiments is indicated in the text. (E) Quiescent T cells were transfected with each of the four individual siRNA duplexes then stimulated for 72 h with PMA/ionomycin. The percentage of cells in each cell‐cycle phase was determined by flow cytometry as described in (D). Source data is available for this figure in the Supplementary Information. Stephen J Orr et al. Mol Syst Biol 2012;8:573 © as stated in the article, figure or figure legend