Figure 3

GSK-3β co-localizes and interacts with Aldolase proteins. (A) HEK293T cells were co-transfected with FLAG-GSK-3β or an empty vector, and with GFP-ALDOC (left), GFP-ALDOB (right) as indicated. Forty eight hours post transfection, cells were harvested and lysates were co-immunoprecipitated with anti-FLAG M2 beads, separated by SDS-PAGE and visualized using the indicated antibodies. (B) HEK293T cells were co-transfected with FLAG-GSK-3β and increasing concentrations of GFP-ALDOC (0-10 mg DNA). Forty eight hours post transfection, cells were harvested and lysates were co-immunoprecipitated with anti-FLAG M2 beads, separated by SDS-PAGE and visualized using the indicated antibodies. Striatin was used as a negative control to rule out non-specific interactions. (C) Mouse brain lysates were co-immunoprecipitated with rabbit anti-GSK-3β antibody or with control un-immuned serum incubated with protein A/G beads and separated by SDS-PAGE. Blots were incubated with anti-GSK-3β, anti-ALDOC or anti-ALDOB antibodies. (D) HEK293T cells grown on cover slips were transfected with GFP-ALDOC (upper panel) or GFP-ALDOB (lower panel). Forty eight hours later, cells were fixed, permeabilized, and incubated with an anti-GSK-3β antibody. Cell nuclei were then stained with DAPI, and viewed using confocal microscopy. (E) Untransfected HEK293T cells grown on cover slips were subjected to immunofluorescence assay as in D, using the indicated antibodies. (F) HeLa cells grown on cover slips were subjected to immunofluorescence assay as in D, using the indicated antibodies.