Figure 3

HDAC3 alters ataxin-7 subcellular localization. (A,B) Expression of ataxin-7 in cytoplasmic and nuclear fractions of HEK293T cells. Cells were transiently transfected with ataxin-7-10Q (wild type), ataxin-7-92Q (expanded) (A), or the 1–266 N-terminal fragment of wild type and expanded ataxin-7 (B) and cotransfected with vector control (v) or HDAC3. Lysates were subjected to subcellular fractionation followed by western blot analysis of nuclear and cytoplasmic fractions. Ataxin-7 antibody revealed the presence of bands corresponding to the wild type, expanded and stop mutant ataxin-7. Bands of higher molecular weight of ataxin-7 are evident when ataxin-7 is co-expressed with HDAC3 (indicated by asterisks) and present in both nuclear and cytoplasmic fractions. Co-transfection of HDAC3 was confirmed by immunoblotting (data not shown). To ensure equal loading, immunoblotting with GAPDH was used for the cytoplasmic fractions and poly-ADP ribose (PARP) for the nuclear fraction. (C) Immunocytochemistry of transfected HEK293T cells. Cells were fixed and stained with antibodies to ataxin-7 conjugated to Alexa 488 (green), HDAC3 conjugated to Alexa 555 (red), and counterstained with DAPI (blue). Only the ataxin-7 and DAPI expression are shown, although co-transfection of HDAC3 was determined by visualization of the red track when appropriate. Cells were imaged using confocal microscopy to elucidate colocalization of ataxin-7 and DAPI. Scale bar represents 5 μm. (D) Quantification of ataxin-7 in nuclear fractions of HEK293T cells. Cells were transiently transfected with ataxin-7-10Q, ataxin-7-92Q and cotransfected with vector control (v) or HDAC3. Lysates were subjected to subcellular fractionation followed by western blot analysis of nuclear fractions. Quantification (n = 3) of ataxin-7 levels were normalized relative to nuclear poly (ADP-ribose) polymerase PARP (1:1000, PARP #9542, Cell Signaling Technology, Boston MA). (E) Cell toxicity determined by Hoescht staining. Cells were transfected with ataxin-7-10Q or expanded ataxin-7-92Q and visualized via immunocytochemistry following addition of Hoescht counterstain.