Figure 2.

Mapping the L-GILZ-binding domains of p53. (a) Schematic illustration of full-length and mutant p53 constructs. TA, transactivation domain; PR, proline-rich region; DBD, DNA-binding domain; OD, oligomerization domain; RD, regulatory domain. (b) p53^−/− HCT116 cells were co-transfected with GST–L-GILZ or GST (in pEBG) together with full-length or mutant p53 constructs. GST–L-GILZ and GST were purified by glutathione–sepharose resin. Co-purified full-length or mutant p53 was detected with an anti-p53 antibody (upper left), and purified GST–L-GILZ or GST was detected by anti-GST antibody (lower left). Lysates were analyzed by Western blot using anti-p53 (upper right) or anti-GST (lower right) antibodies to control for plasmid expression. (c) Complex formation was measured by in situ PLA in p53^−/− HCT116 cells transiently transfected with L-GILZ together with full-length or mutant p53. Primary antibodies were goat anti-GILZ and rabbit anti-p53. Secondary antibodies were anti-goat PLA-plus probe for L-GILZ and anti-rabbit PLA-minus probe for p53. In situ PLA is indicated by red signals from rolling cycle amplification products. Positive cells are indicated by arrows. Scale bar represents 30 μm. Images are representative of three experiments with consistent results