Figure 2. AIP interacts with multiple proteins from the HSP90 and HSP70 families of molecular chaperones.

WT AIP interactions with HSP90AA1 (A) and HSP90AB1 (B) were confirmed by co-IP, validating the experimental procedures. Also by co-IP, HSPA8 interacted with WT AIP (C), but not with the p.R304* mutant (D), as predicted by the pull-down experiment results. Interactions with two novel molecular chaperones were validated by co-IP: HSPA5 (E) and HSPA9 (F). Co-localization of AIP and HSPA9 (G) was confirmed in the mitochondrial network, with a Pearson’s R-value of 0.72. (H) AIP does not co-immunoprecipitate with PRKACA, ruling out direct interaction of these two proteins. (I) However, in the presence of HSP90AB1, the three proteins co-immunoprecipitate with the anti-Myc, anti-HA and anti-Flag antibodies. (J) Areas of co-localization for AIP and PRKACA were detected in the cytoplasm, although with a weaker correlation compared with HSPA9, for a Pearson’s R-value of 0.39. For the co-IP experiments a-f and h, the left panels represent anti-Myc western blot (WB) membranes and the right panels, anti-HA WB membranes. For the co-IP experiment presented in i the additional panel at the extreme right presents an anti-Flag WB membrane. The blue arrows in all the panels represent the protein of interest in each WB membrane (Myc-AIP on the left panels, and HA-tagged proteins on the right panels, plus Flag-PRKACA in experiment i). The arrowheads in all the panels point out the heavy (top) and light (bottom) chains of mouse immunoglobulins. IP: immunoprecipitation, MWM: molecular weight marker. The immunocytofluorescence images (G and J) are reconstructions of representative images of the z-stacks obtained, at a 63× magnification. Top left: nuclei (DAPI), top right: HSPA9 (G) or PRKACA (J), bottom left: AIP and bottom right: merged image. Inserts in the merged images present 2-D intensity histograms corresponding to the co-localization calculations.