Total protein was extracted from cells using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech). Equivalent protein from different samples was separated by 10% SDS-PAGE protein electrophoresis, following by transformation onto PVDF membranes (Merck Millipore, Billerica, MA). The membranes were blocked with 5% skim milk at room temperature. After that, the membranes were incubated with primary antibodies, p53 (1:1000, Proteintech, Wuhan, China), EBNA2 (1:500, Abcam, Cambridge, MA), LMP1 (1:500, Abcam), EBNA3A (1:2000, Abcam), EBNA3C (1:1000, Eterlifer, Birmingham, UK), p65 (1:1000, Proteintech), p-p65 (1:1000, Abcam), p52/p100 (1:500, Proteintech), c-MYC (1:1000, Proteintech), cyclin E (1:250, Santa Cruz Biotechnology, Santa Cruz, CA), BZLF1 (1:500, Santa Cruz Biotechnology), BMRF1 (1:3000, Merck Millipore) or p18 (1:2000, Thermo Fisher Scientific) at 4 °C overnight. After the membranes were washed with TBST for several times, secondary antibodies labelled with horseradish peroxidase were incubated with the membranes. β-actin antibody (1:2000, Proteintech) was used as a reference protein for normalization. The grey level of the protein bands was examined by Image J software (Bethesda, MD).
Ebna2
Manufactured by Abcam
Sourced in United States
EBNA2 is a nuclear phosphoprotein that is essential for the immortalization of B lymphocytes by the Epstein-Barr virus (EBV). It functions as a transcriptional activator and plays a crucial role in the regulation of viral and cellular gene expression during EBV infection.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
β actin antibody, by Proteintech (1 mentions)
Tissue or cell total protein extraction kit, by Sangon (1 mentions)
Ebna3a, by Abcam (1 mentions)
Bzlf1, by Santa Cruz Biotechnology (1 mentions)
Cyclin e, by Santa Cruz Biotechnology (1 mentions)
C myc, by Proteintech (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Parp1, by Active Motif (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Ebna1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse igg, by GE Healthcare (1 mentions)
Lmp2a, by Abcam (1 mentions)
Ebna1, by Abcam (1 mentions)
4 protocols using ebna2
1
Western Blot Analysis of Viral and Cellular Proteins
2
Western Blot Analysis of Protein Extracts
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Cell lysates were prepared in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA; Millipore) supplemented with 1× protease inhibitor cocktail (Thermo Scientific). Protein extracts were obtained by centrifugation at 3,000 × g for 10 min at 4°C. Protein concentration was measured using a bicinchoninic acid (BCA) protein assay (Pierce). Lysates were boiled with 1× Laemmli sample buffer (Bio-Rad) containing 1.25% β-mercaptoethanol (Sigma-Aldrich). Proteins were resolved by gel electrophoresis on a 4 to 20% polyacrylamide gradient Mini-Protean TGX precast gel (Bio-Rad) and transferred to an Immobilon-P membrane (Millipore). Membranes were blocked in 5% milk in PBS-T for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against PARP1 (Active Motif), EBNA2 (Abcam), LMP1 (Abcam), and actin (Sigma-Aldrich).
3
Immunoblotting analysis of EBV proteins
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Proteins were extracted from cells using radioimmunoprecipitation buffer. Twenty µg of total proteins were subjected to SDS-PAGE and transferred onto PVDF membrane (GE Healthcare, Chicago, IL, USA). Membranes were then probed with primary antibodies for EBV proteins, including EBNA1 (Santa Cruz Biotech, Santa Cruz, CA, USA), EBNA2 (Abcam, Cambridge, UK), LMP1 (Dako, Glostrup, Denmark), LMP2A (Santa Cruz Biotech, Santa Cruz, CA, USA) and β-actin (A1978; Sigma, St. Louis, MO, USA). After probing, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or HRP-conjugated anti-sheep IgG (GE Healthcare, Chicago, IL, USA) and visualized using Image Quant LAS 4000mini (GE Healthcare, Chicago, IL, USA).
4
EBV Latency and Lytic Expression
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Formalin-fixed paraffin-embedded sections were used for all stainings. Slides were immunostained automatically (DAKO, Carpenteria, CA, USA) according to manufacturers' protocol. All antibodies were purchased from DAKO unless stated otherwise (Supplemental list). Immunostains were performed on a case-by-case basis as required for establishing a diagnosis according to the 2017 Revised WHO classification. The presence of EBV was determined by EBER and performed as previously reported [32] . All EBER-positive biopsies with sufficient material were stained for EBV viral protein expression: latency membrane protein (LMP1), LMP2A, EBNA1, EBNA2 (Abcam, Cambridge Science Park, Boston, USA), and ZEBRA (BZ1). Based on the expression of Epstein-Barr viral LMP and nuclear antigen 2 (EBNA2), we defined three different conventional latency types of EBV infection. LMP1 -/EBNA2-, LMP1+/EBNA2-, and LMP1+/EBNA2+ represent latency types I, II, and III (respectively also referred to as restricted, intermediate and broad latency). Occurrence of lytic viral replication was determined by ZEBRA staining. The number of ZEBRA-positive cells was determined based on the range of ZEBRA-positive nuclei in 5 high power fields (HPFs) (×400) as previously described [33] .
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