Interaction of the S100A14 protein with actin and analyses of its functional interaction with S100A16. A, An extract of MCF7 cells was immunoprecipitated (IP) with control rabbit IgG, anti-S100A14 or anti-actin antibodies. Precipitated and co-precipitated proteins were detected by immunoblotting with anti-S100A14 and anti-actin antibodies. The following were added to the extracts prior to precipitation. CaCl2; 0 mM (lane 1), 0.5 mM (lane 2) or 2 mM (lane 3), or, 2 mM CaCl2 and 10 mM EDTA (lane 4). Arrows indicate actin and S100A14. B, Co-localization of S100A14 with polymerized actin and S100A16 in MCF7 cells. Co-immunostaining with the anti-S100A14 antibody and a FITC-labeled second antibody (green), and with rhodamine-conjugated phalloidin (red) showed colocalization of both S100A14 and actin along the cell membrane of MCF7 cells (top). Co-localization of S100A14 and S100A16 was also confirmed in MCF7 cells (bottom). Scale bars; 20 μm. C, Analysis of the mRNA expression of S100A14 and S100A16 in MCF7 cells transfected with siRNA for S100A14 or S100A16. Gene knockdown of S100A14 or S100A16 does not affect the mRNA expression level of the other S100 protein in MCF7 cells. The relative mRNA expression levels were normalized to the GAPDH mRNA expression level. Error bars, + SD, n=3. D, Effect of siRNA targeted against S100A14 or S100A16 on the expression of each protein. Western blots of extracts of MCF7 cells transfected with siRNA targeted against S100A14 or S100A16 were probed with antibodies against S100A14, S100A16 and b-actin. E, Expression of S100A14 or S100A16 protein following knockdown of the counterpart molecule. Immunofluorescence analysis of the protein expression and localization of S100A14 and S100A16 in MCF7 cells 48 h after transfection with siRNA targeted against each protein. Scale bars; 10 μm.