Fig. 4
S100A14 interacts with kidney-type glutaminase in cultures of HCC cells. (A) Schematic of the experimental workflow to identify binding partners of S100A14 in Huh7 cells. (B) A list of representative proteins that co-immunoprecipitated with S100A14. (C and D) Immunoprecipitates were obtained as described from panel A in Huh7 and Hep3B cells, then analyzed using a pan-antibody against glutaminases (GLS) or antibodies specifically against the kidney-type isoform (GLSKGA) or isoform C (GLSGAC). (E and F) Cell lysates were immunoprecipitated using a pan-antibody against GLS or an IgG control antibody, then immunoblotted for S100A14. (G, H, I and J) Huh7 and Hep3B cells were examined using confocal fluorescence microscopy to observe interactions between (G and H) endogenous S100A14 and GLSKGA or between (I and J) FLAG-tagged S100A14 and HA-tagged GLSKGA expressed from transfected plasmids. Mitochondria were detected using MitoTracker Red CMXRos. Quantitation of fluorescence intensity tracings (along the white arrows) in “Merge” images was shown at the far right. n = 3. Scale bar, 10 μm