Supplemental material Figure S10.
FIG. S10. Validation of EBV gene expression. To confirm EBV gene expression at the protein level as well as at the mRNA level, immunoblotting was performed with antibodies against EBNA1, EBNA2, LMP1, BZLF1 and BHRF1 of (A) Akata, P493-6 (+c-myc, -tetracycline) and P493-6 (-c-myc, 24h after addition of 0.1 µg/ml tetracycline), as well as of (B) EREB2-5 0 hour (-EBNA2), 3h, 8h, 48h and 8 day (+EBNA2) and LCL 1.25 cellular extracts. Gel loading is indicated by Ponceau red staining. High expression of EBNA1 is presumably selected for in P493-6 cells as EBNA1 is required for the episomal replication of the “tet-off” c-myc expression plasmid. The high molecular weight of EBNA2 is due to the fusion with the estrogen receptor and is regulated at the posttranslational level. BHRF1 is presumably expressed from a spliced BamHI-W-H fusion transcript in P493-6 as well as EREB2-5 cells (1). BZLF1 has been described to be regulated at the posttranscriptional level by antisense transcription (2, 3). (C) Because the LMP2A expression on the lymphochip did not follow precisely the expected pattern, LMP2A mRNA expression was quantified by real-time PCR. RNA was reverse transcribed into cDNA from total RNA of Akata, P493-6 (+c-myc, -tetracycline), P493-6 (-c-myc, 24h after addition of 0.1 µg/ml tetracycline), EREB2-5 0h (-EBNA2), 4h, 8h, and 24h (+EBNA2) and LCL 1.25. The given values are normalized to the expression of the reference gene RPL23A.
MATERIALS AND METHODS RELATED TO THE FIGURE S10
Immunoblotting Analysis
The antibodies used were monoclonal antibodies anti-LMP1 (CS1-4), anti-EBNA1 (R3.1.3), anti-EBNA2 (1H4), anti-BZLF1 (BZ1), and anti-BHRF1 (5G2-2). Equal loading is controlled by Ponceau red staining.
Real-time Quantitative RT-PCR Analysis
RNAs were isolated according to the RNeasy manufacturer protocol (Qiagen). The cDNA synthesis reaction was performed according to the Superscript 2 RT manufacturer protocol (Invitrogen). After dilution of cDNA samples (1:10 with H2O), real-time quantitative RT-PCR was performed with the LightCycler 480 II Instrument using the LightCycler480 Sybr Green Master Kit (Roche Applied Science). The experiment included a standard curve for the target gene LMP2A (primers: 5'-CTAATCGCTGGTGGCAGTATTTTG-3', and 5'-ACCGAGGCACATAAAAACTGGAC-3') and the reference gene RPL23A (primer: 5’-ctcctaaagctgaagccaaa-3’, and 5’-gcctgtttaatctggtgctt-3’) in triplicates over 4 orders of magnitude. The samples were run in duplicates. Relative quantification of LMP2A expression was done including efficiency correction via relative standard curves.
REFERENCES RELATED TO THE FIGURE S10
1. Kelly, G. L., H. M. Long, J. Stylianou, W. A. Thomas, A. Leese, A. I. Bell, G. W. Bornkamm, J. Mautner, A. B. Rickinson, and M. Rowe. 2009. An Epstein-Barr virus anti-apoptotic protein constitutively expressed in transformed cells and implicated in Burkitt lymphomagenesis: the BamHI W promoter/ BHRF1 connection. PLOS Biol. In press.
2. Prang, N., H. Wolf, and F. Schwarzmann. 1995. Epstein-Barr virus lytic replication is controlled by posttranscriptional negative regulation of BZLF1. J Virol. 69:2644-2648.
3. Prang, N., H. Wolf, and F. Schwarzmann. 1999. Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1. J Med Virol. 59:512-519.