Figure 3. Loss of the RBR alters the subnuclear distribution of SCML2A.

(A and B) Salt fractionation of 293T-REx induced to express SCML2A WT (A) and ΔRBR (B) with increasing amounts of doxycycline (dox). The distribution of RNAPIIA and RNAPIIO are shown as fractionation and loading control. (C) Densitometry-based quantification of the ratio of nucleoplasmic vs chromatin-associated SCML2A WT (black bars) and ΔRBR (white bars). Bars show mean measurements from two independent clones at increasing levels of inductions. p-values were calculated with 2-way ANOVA. (D) Salt fractionation of 293T-REx induced to express SCML2A WT, ΔRBR, and the MBT-domain mutant D182Q. (E and F) Fluorescence microscopy of HeLa cells transiently transfected with vectors encoding GFP-SCML2A_WT (E) or GFP-SCML2A_ΔRBR (F). (G) Quantification of SCML2 spots in multiple cells transfected as in (E) and (F). Bars represent mean + SEM. N = 11 for SCML2A; N = 17 for SCML2A_ΔRBR. p-value was calculated with an unpaired t test.

DOI: http://dx.doi.org/10.7554/eLife.02637.007

Figure 3—figure supplement 1. Nuclear distribution of SCML2A_D182A and SCML2B.

(A) Domain organization of SCML2A and SCML2B. (B) Salt fractionation experiment as in Figure 3D but using a different mutation (ala instead than gln) in the MBT domain. (C and D) Salt fractionation of 293T-REx induced to express SCML2B WT (A) and ΔRBR (B) with increasing amounts of doxycycline (dox). The distribution of RNAPIIA and RNAPIIO are shown as fractionation and loading control. (E) Densitometry-based quantification of the ratio of nucleoplasmic vs chromatin-associated SCML2B WT (black bars) and ΔRBR (white bars). Bars show mean measurements from two independent clones at increasing levels of inductions. (F and G) Fluorescence microscopy of HeLa cells transiently transfected with vectors encoding GFP-SCML2B (F) or GFP-SCML2B_ΔRBR (G). (H) Immunofluorescence microscopy of endogenous SCML2 in HeLa cells using affinity-purified SCML2 antibody.

DOI: http://dx.doi.org/10.7554/eLife.02637.008