The intensity of E84 staining is correlated with the PRMT1 protein level. (A) E84 staining of 293T cells. One million control 293T cells or 293T cells that overexpress PRMT1 (isoforms V1 or V2) were incubated in 10 nM E84 in PBS for 30 minutes and then subjected to FACS analysis. Mean fluorescence intensity (MFI) values are indicated in a plot representative of 3 independent experiments. Western blots for detecting PRMT1 in 293T cells are to the right. PRMT1 was normalized to tubulin, and ratios are presented below the PRMT1 blot. (B) MV4-11 cells that express control short hairpin RNA or shPRMT1 were stained with E84 (left panel) and anti-PRMT1 antibody (middle panel); western blots of PRMT1 (right panel) show the efficiency of PRMT1 knockdown. (C) E84 staining of 6133 cells correlates with endogenous PRMT1 expression. Left panel: 6133 cells were stained with E84 and analyzed by FACS. Middle panel: PRMT1 protein expression levels in E84-high and E84-low cells. The 6133 cells were sorted into 2 populations based on E84 staining intensity for western blotting with an anti-PRMT1 antibody. Tubulin was used as a loading control. Triangles indicate increasing amounts of lysate loaded. Right panel: mRNA levels of PRMT1, PRMT3, and PRMT6 in E84-high and E84-low cells were measured using real-time PCR assays. The data are shown as mean ± standard deviation. *P < .05. (D) E84 staining of 6133 cells expressing PRMT1 from a doxycycline-inducible promoter. Left panel: contour plots gated according to E84 staining. Right panels: E84 histogram plots of 6133 cell lines. MFI values are in the plots. Representative results from at least 3 independent experiments are presented. Western blots measure the PRMT1 protein levels in 6133 cells with and without doxycycline induction. The relative PRMT1 protein levels were quantitated and normalized to tubulin protein. ns, not significant.