Fig. 1: GBP1 and GBP2 specifically interact with MCL-1.
From: Guanylate-binding proteins induce apoptosis of leukemia cells by regulating MCL-1 and BAK
a Yeast growth was demonstrated in colonies expressing both GBP2 and MCL-1 fused to the GAL4 DNA activation domain and binding domain, respectively. b, c The endogenous protein–protein interaction between GBP2 and MCL-1 was confirmed using immunoprecipitations followed by western blot analyses using indicated antibodies in K562 cells (b) and HL-60 cells (c). IgG was used as an immunoprecipitation control. d In vitro binding of GBP2 and MCL-1 was determined using purified recombinant proteins (0.5 µg) of GBP2 and MCL-1 with or without the presence SAHBB (10 µM), a BH3-specific inhibitor of MCL-1. Subsequently, immunoprecipitation and immunoblotting were performed with indicated antibodies. e The interaction of GBP2 with prosurvival members of the BCL-2 family was examined by immunoprecipitation following cotransfection of 3 µg of plasmids encoding HA-GBP2 and FLAG-BCL-2 family members in K562 cells. Arrows indicate the expected positions of respective proteins. GAPDH was included as a loading control. f, g Schematic representations of the plasmids encoding WT and mutants of MCL-1 (f) and GBP2 (g), which were generated to determine the binding domains. 293 T cells (2 × 106) were cotransfected with indicated plasmids. After transfection for 24 h, cell lysates were immunoprecipitated with anti-HA (f) or anti-FLAG antibodies (g) followed by immunoblotting with the indicated antibodies. h The interaction between GBP1 and MCL-1 was assessed in K562 cells after the transfection of plasmids (3 µg) encoding GBP1 or GBP2 followed by immunoprecipitation. i The intracellular localization and colocalization of endogenous MCL-1 with GBP1 or GBP2 in K562 cells were visualized by fluorescent confocal microscopy. For all immunoblot images presented throughout this manuscript, the membrane was cut into pieces according to the estimated molecular weight of proteins of interest and probed with the indicated antibodies. All cropped blots were run under the same experimental conditions.
