Figure 7.

Immunofluorescence analysis of the intracellular location of extracellularly added GAL3BP. a, H4-APPsw cells were incubated with extracellularly applied recombinant GAL3BP (5 μg/ml) for 24 h and then stained with anti-GAL3BP (c-Myc) antibody. Arrowheads, intracellular signals of recombinant GAL3BP. Vehicle treatment was used as negative control (N.C.). Scale bars, 50 μm. b, H4-APPsw cells treated with GAL3BP were co-stained for GAL3BP (c-Myc), the early endosome marker EEA1, and nuclei (DAPI). Arrowheads, co-localization of intracellular signals of recombinant GAL3BP and EEA1. Scale bars, 20 μm. c, H4-APPsw cells were pre-incubated with or without nystatin (50 μg/ml) for 1 h and then incubated with recombinant GAL3BP for 3 h. The cells were stained for GAL3BP (red) and DAPI (blue). Asterisks, cells with no signal for intracellular GAL3BP. Arrowheads, intracellular dotlike signals of recombinant GAL3BP. Arrows, large grainlike signals for GAL3BP. Scale bars, 20 μm. d, quantification of intracellular signals for GAL3BP. Cells were classified into three groups: no intracellular signals, intracellular dotlike signals, and large grainlike signals (corresponding to asterisks, arrowheads, and arrows in c, respectively). More than 500 cells were counted in randomly taken fields and statistically analyzed using the χ² test. e and f, H4-APPsw cells were treated with the recombinant GAL3BP (c-Myc–tagged). The cells were co-stained for APP, EEA1, and nuclei (DAPI) (e). Arrowheads, co-localization of intracellular signals of APP and EEA1 (e). The cells were also subjected to a PLA using antibodies to APP and c-Myc (f). Red signals indicate the co-localization of intracellular signals of APP and recombinant GAL3BP (f). Scale bars, 20 μm.