Fig. 5.

Interaction between IGFBP5 and retinoic acid receptors. A Cell lysates were immunoprecipitated with either anti-RXRα or anti-RARα antibodies and then subjected to western blot analysis using anti-IGFBP5 antibody. Reciprocal immunoprecipitation (IP) was performed with anti-IGFBP5 antibody, followed by western blots (WB) using either anti-RXRα or anti-RARα antibodies. P and Sup refer to the pellet and supernatant, respectively, of the immunoprecipitation reaction. B Representative images of the Duolink assay to identify the physical proximity between IGFBP5 and RARα. The red staining indicates a close interaction between IGFBP5 and RARα ten minutes after MSCs were incubated with His-tagged IGFBP5. The nuclei were stained with DAPI (blue). The scale bar corresponds to 100 microns. C Recombinant RARα, immobilized on protein A beads, was incubated with IGFBP5 in the presence or absence of ATRA, followed by western blot analysis using anti-IGFBP5 antibody. P and Sup refer to the pellet and supernatant, respectively, of the reaction. D The graph shows the percentage of senescent cells following incubation with IGFBP5 under different experimental conditions. The symbol *** p < 0.001 indicates statistical significance between untreated cells and samples incubated with IGFBP5 (first and second column). The symbols ### p < 0.001 and ## p < 0.01 indicate the statistically significance between the sample treated with IGFBP5 (second column from left), which was chosen as reference, and the others with IGFBP5 in combination with further treatments. E Fluorescence quenching assay. The graphs show the ultraviolet peak emission of IGFBP5 and RARα, due to tryptophan, phenylalanine, and tyrosine, either in the absence or presence of increasing amounts of ATRA, which acted as the quencher. Data are reported with standard deviation. The symbols * p < 0.05, ** p < 0.01 indicate statistical significance between samples incubated with and without ATRA. The latter condition was chosen as the reference