FIGURE 4.
Essential role of Rictor in mRNA translation of type II ISGs. A, serum-starved Rictor+/+ and Rictor−/− MEFs were either left untreated or treated with mouse IFNγ. Cell lysates were bound to the biotin labeled cap analog, m7GTP, and to streptavidin beads (39). After extensive washing, bound proteins were resolved by SDS-PAGE and immunoblotted with antibodies against eIF4G, eIF4A, eIF4E, and 4E-BP1. The cell lysates used for this experiment were from the same experiment shown in Fig. 1D. B, Rictor+/+ and Rictor−/− MEFs were either left untreated or treated with mouse IFNγ in DMEM containing 0.5% FBS for 24 h. Cell lysates were layered on 5–50% sucrose gradients and subjected to density gradient centrifugation, and then fractions were collected by continuous monitoring of absorbance at 254 nm. Absorbance at 254 nm is shown as a function of gradient depth, and the 40S, 60S, and 80S peaks and polysomal fractions are indicated. C and D, polysomal fractions were pooled, and RNA was isolated. Subsequently, quantitative real-time PCR was carried out to determine CxCl10 (C) and Irf9 (D) mRNA expression in polysomal fractions, using Gapdh for normalization. Data are expressed as -fold change over control untreated cells, and error bars represent mean ± S.E. of five independent experiments, including the ones shown in B. Statistical analyses were performed using Student's t test. *, p < 0.05.