Figure 2

Co-knockdown of Zn72D and bel restores MSL localization and X-linked gene expression. (A) GFP-MSL2 expressing S2 cells were either untreated or treated with Zn72D, bel, or Zn72D+bel dsRNAs. The left panels show GFP fluorescence (green), the middle panels show DAPI staining to highlight the nucleus (blue), and right panels show the merged image. (B) Western blots with anti-Zn72D (top), anti-Bel (middle), or anti-γ-tubulin (bottom) as a loading control, in knockdowns indicated above. (C) qRT-PCR for the X-linked genes arm, CG14804, and mRpL16 and the autosomal gene RpII140 was done after S2 cells were untreated (gray bars), or treated with dsRNAs to knockdown mle (white), Zn72D (black), bel (blue), or Zn72D+bel (red). Samples were normalized first to rp49 and then to the untreated sample, setting untreated to 1. The error bars represent the average of three independent experiments, with qPCR performed in triplicate, and error was determined using standard error propagation methods. (D) Zn72D and Bel are both present in the cytoplasm. Cells were stained with antibodies to HA (red), Bel (green), and DAPI (blue) to delineate the nucleus. The nucleolar staining in the anti-Bel image was the result of antibody cross-reactivity, as when bel is knocked down, the nucleolar staining remained while the Bel staining decreased (data not shown). (E) Zn72D and Bel are present in both nuclear and cytoplasmic extracts. The cytoplasmic extract is enriched for β-tubulin and depleted for H3K4me3, whereas the nuclear extract is depleted for β-tubulin and enriched for H3K4me3.