Fig. 6

NPM binds to TLR4. a Representative image of WB analysis of co-immunoprecipitation between NPM and TLR4 in hCmPCs. Cells were immunoprecipitated with NPM antibody (Ip-NPM) or an irrelevant negative antibody rabbit IgG (IP-IgG). Western blotting with TLR4 revealed that NPM interacts with TLR4 in hCmPCs. The efficiency of immunoprecipitation was assessed with an NPM antibody. One-twentieth of the immunoprecipitated whole-cell extract (Input) was loaded as a reference. Uncropped images of blots are shown in Additional file 1: Figure S1c. b Proximity ligation assay of NPM and TLR4 interaction analysis in serum starved hCmPCs treated with rNPM (0.5μg/ml) for 8h. Lower panels represent zoomed in images of the original PLA images. Red dots indicate that even under physiological conditions, NPM interacts with TLR4. When rNPM is administered, the rate of interaction increases. As a negative control, the IgG specific for each antibody (i.e. normal rabbit IgG used as the control of TLR4 antibody) and normal mouse IgG for NPM antibody (left panel) revealed the specificity of NPM and TLR4 interaction, since no red dots were present in the PLA. Scale bar 10μm. c Bar graph of the quantification of dots per nuclei (n = 5; ^*P <0.05)