Fig. 4
EDAL downregulates H3K27me3 level by causing the degradation of EZH2. a EDAL, reverse EDAL (revEDAL), XLOC_023040, ENSMUSG00000087590.2 (ENS_87590.2), or XLOC_059122 was overexpressed in N2a cells for 48 h and then EZH2, H3K4me3, H3K27me3, and K3K79me3 levels were measured by Western blotting. The plasmid pCAGGS-eGFP containing a HA tag was used as a transfection control. b N2a cells were transfected with pcDNA3.1, pcDNA-EDAL, pcDNA-revEDAL, pcNDA-XLOC_023040, pcDNA-ENS_87590.2, or pcDNA-XLOC_059122, and pCAGGS-EZH2-FLAG and pCAGGS-eGFP-HA (transfection control). EZH2-FLAG levels were measured by Western blotting and normalized to H3. c N2a cells were infected with rRABV, rRABV-EDAL, rRABV-revEDAL, rRABV-XLOC_023040, rRABV-ENS_87590.2, or rRABV-XLOC_059122 at MOI 3. At 36 hpi, the EZH2 and H3K27me3 levels were resolved by Western blotting and normalized to H3. d N2a cells were transfected with siEDAL or siNC (negative control) for 8 h and then transfected with pcDNA3.1 or pcDNA-EDAL. Then EZH2 and H3K27me3 levels were resolved by Western blotting and normalized to H3. e N2a cells were transfected with pcDNA3.1, pcDNA-EDAL, pcDNA-revEDAL, pcNDA-XLOC_023040, pcDNA-ENS_87590.2, or pcDNA-XLOC_059122. The mRNA levels of EZH2 were analyzed by qPCR (n = 3). f pcDNA3.1, pcDNA-EDAL, or pcDNA-revEDAL was transfected into N2a cells. The specific inhibitors for proteasome and lysosome, MG132 (10 μM) and NH4Cl (5 mM), were applied. Then EZH2 and H3K27me3 levels were analyzed by Western blotting and normalized to H3. g pcDNA3.1, pcDNA-EDAL, or pcDNA-revEDAL was transfected together with pCAGGS-EZH2-flag into N2a cells. The specific inhibitors for proteasome and lysosome, MG132 (10 μM) and NH4Cl (5 mM), were applied. Then EZH2-flag level was analyzed by Western blotting and normalized to H3. h pcDNA3.1, pcDNA-EDAL, or pcDNA-revEDAL was transfected together with pCAGGS-EZH2-flag into N2a cells. At 36 h post transfection, EZH2-flag and LAMP-1 were analyzed by immunofluorescence. Scale bar, 5 μm. Western blot data are representative of at least two independent experiments