To confirm that successful inhibition of EZH2 was achieved by DZNep in mice, levels of EZH2 and H3K27me3 in splenocytes were examined using Western blot. Twenty μg of protein was separated by SDS-PAGE before electroblotting onto a nitrocellulose membrane. Blots were probed with anti-EZH2 or anti-H3K27me3 antibodies (both from Cell Signaling). B-actin (Sigma-Aldrich) and H3 (Cell Signaling) were used as loading controls. Since we previously showed that JAM-A is regulated by EZH2 and downregulated by DZNep in lupus CD4+ T cells (6 (link)), we also examined levels of JAM-A (Santa Cruz Biotechnology) in these cells. Densitometry was analyzed using ImageJ [27].
Protocol detail
Validating EZH2 Inhibition by DZNep
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Actin
Anti antibodies
Cd4 t cells
Cells
Densitometry
Dznep
Ezh2
Inhibition
Jam a
Lupus
Membrane
Mice
Nitrocellulose
Protein
Sds page
Western blot
Corresponding Organization : University of Pittsburgh
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Variable analysis
independent variables
- DZNep treatment
- Levels of EZH2
- Levels of H3K27me3
- Levels of JAM-A
- Protein loading (20 μg)
- B-actin as loading control
- H3 as loading control
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