Figure 2

EZH2β is expressed in a variety of adult human tissues. (A) Analysis of EZH2β, EZH2α, as well as associated complex co-factors SUZ12, EED, RBBP4 and RBBP7 transcripts in 22 human tissues. RT-PCR was performed with primers designed to differentiate the two EZH2 isoforms. Analysis of glyceraldehyde-3-phosphate dehydrogenase was used as amplification control. Red box indicates tissues that possess highly proliferative capacity and preferential co-expression of all PRC2 complex transcripts, including EZH2β. (B) Specificity of EZH2α and EZH2β antibodies. Isoform-specific antibodies were tested using western blot of untransfected pancreatic cancer cell lines (Additional file1: Figure S1) and also shown here in cells transfected with histidine (HIS) epitope-tagged EZH2α, EZH2β or both isoforms (EZH2αβ). Labeling of the HIS-tag was used as loading control. (C) Tissue distribution of EZH2β at the protein level where the presence of EZH2β was determined by western blot analysis of human tissues with the EZH2β-specific antibody. β-actin was used as loading control. (D) Fluorescent immunohistochemistry of samples of frozen human testis was performed by laser confocal microscopy, in sections labeled for EZH2β (left) and total EZH2 (right). The white circle encompasses primary and secondary spermatocytes, whereas primary spermatogonia are immediately adjacent to the basal lamina (white arrow). Nuclei are counterstained with Hoechst. Images were obtained at 10× magnification. White scale bar represents 100 μm. Together, these results validate that the two major isoforms generated by the human EZH2 locus, namely EZH2α and EZH2β, are translated into proteins that can be detected not only in cell lysates but also in whole tissues. EED: embryonic ectoderm development; EZH2: enhancer of zeste homologue 2; GADPH: glyceraldehyde-3-phosphate dehydrogenase; HIS: histidine; RBBP: retinoblastoma binding protein; SUZ12: suppressor of zeste 12.