Role of the Akt pathway in mRNA translation of interferon-stimulated genes

Kaur et al. 10.1073/pnas.0710907105.

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Materials and Methods

Cell lines and Reagents.Human recombinant IFN-a was provided by Hoffman La Roche, Recombinant mouse IFN-b was provided by Biogen Idec. Recombinant mouse IFN-g was from PBL Biomedical Laboratories. U2OS cells were grown in McCoy medium supplemented with 10% FCS and antibiotics. A monoclonal anti-human ISG15 antibody was a kind gift from Ernest Bordon (Taussig Cancer Center, Cleveland). A rabbit polyclonal antibody against mouse ISG15 was kindly provided by Dong-Er Zhang (Scripps Research Institute, La Jolla, CA). A rabbit polyclonal antibody against an epitope comprising residues 105-119 (CLKKKVSQREQVHEDQ) of mouse ISG15 was generated with New England Peptides and used in some experiments. Antibodies against Akt, p70 S6K, rpS6, 4E-BP1 and their phosphorylated forms were from Cell Signaling Technologies. An anti-p70 S6K antibody was purchased from Santa Cruz Biotechnology. Anti-phospho-Ser-727 Stat1 and anti-Stat1 antibodies were from Upstate Biotechnology. An antiphospho-Tyr-701 Stat1 antibody was from Cell Signaling. Antibodies against tubulin and IP10 were from Abcam.

Cell Lysis and Immunoblotting. Cells were treated with the indicated IFNs for the indicated times and lysed in phosphorylation lysis buffer (PLB) as described (1, 2). Equal protein aliquots were resolved by SDS/PAGE, and immunoblotting using an ECL method was performed as in our previous studies (2-4).

Luciferase Reporter Assays. The ISRE luciferase construct containing the wild-type ISG15 ISRE was from Richard Pine (Public Health Research Institute, New York). The 8X GAS construct was from Christopher Glass (University of California, San Diego).

Quantitative RT-PCR. Cells were treated with 5 ´ 103 units/ml of IFN-a or 2.5 ´ 103 units/ml of IFN-g for 6 h, and RNA was isolated with a RNeasy kit (Qiagen). One microgram of total RNA was reverse-transcribed into cDNA by using the Omniscript RT kit and oligo(dT) primer (Qiagen). Real-time PCR for Isg15 and Ip10 genes was then carried out by using commercially available FAM-labeled probes and primers (Applied Biosystems) and ABI7900 Sequence detection system (Applied Biosystems). Relative quantitation of mRNA levels was plotted as fold increase as compared with untreated samples. GAPDH was used for normalization (5). DCt values (target gene Ct minus GAPDH Ct) for each sample were averaged, and DDCt was calculated as described (3). mRNA amplification was determined by formula 2-DDCt (5).

Isolation of Polysomal RNA. Akt WT and Akt 1-/-2-/- MEFS were treated with mIFN-a for 40 hours, and cells were harvested in PBS containing 100 mg/ml of cycloheximide. Polysomal fractionation was performed by using slight modifications of previously described protocols (6, 7). Cell pellets were suspended in hypotonic lysis buffer [5 mM Tris (pH 7.5), 2.5 mM MgCl2, 1.5 mM KCl] supplemented with protease inhibitors (Calbiochem) and RNase-In (Ambion), 1 mM DTT, and 100 mg/ml of Cycloheximide. Triton X-100 and sodium deoxycholate were added to the lysates to a final concentration of 0.5% each. The lysates were clarified at 12,000 ´ g for 5 min, and supernatants were layered over 10-50% continuous sucrose gradient in 20 mM Hepes (pH 7.6), 100 mM KCl, 5 mM MgCl2, plus protease and RNase inhibitors. After ultracentrifugation for 1 h and 50 min, fractions were collected, monitoring the absorbance at 254/260 nm as a function of gradient depth. The polysomal fractions were pooled, and total RNA from polysomal fractions was isolated with a Rnaqeous-micro kit from Ambion. Total polysomal RNA was reverse-transcribed into cDNA using the Omniscript RT kit and oligo(dT) primer (Qiagen).

1. Lekmine F, et al. (2003) Activation of the p70 S6 kinase and phosphorylation of the 4E-BP1 repressor of mRNA translation by type I interferons. J Biol Chem 278:27772-27780.

2. Lekmine F, et al. (2004) Interferon-g engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein. Exp Cell Res 295:173-182.

3. Kaur S, et al. (2007) Regulatory effects of mammalian target of rapamycin-activated pathways in type I and II interferon signaling. J Biol Chem 282:1757-1768.

4. Kaur S, et al. (2005) Role of protein kinase C-d (PKC-d) in the generation of the effects of IFN-a in chronic myelogenous leukemia cells. Exp Hematol 33:550-557.

5. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402-408.

6. Koritzinsky M, et al. (2006) Gene expression during acute and prolonged hypoxia is regulated by distinct mechanisms of translational control. EMBO J 25:1114-1125.

7. Arava Y (2003) Isolation of polysomal RNA for microarray analysis. Methods Mol Biol 224:79-87.

This Article

  1. Published online before print March 13, 2008, doi: 10.1073/pnas.0710907105 PNAS March 25, 2008 vol. 105 no. 12 4808-4813
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