Expression of BAT3 in J774A.1 cells and bone marrow-derived macrophages (BMDM) in response to non-lethal heat shock.

<p>A. J774A.1 cells and BMDM were subjected to non-lethal heat shock at 42°C for 10 minutes and rested for 1 hour. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on SDS-PAGE gels. Following electrophoresis and western blotting, the blots were developed using a rabbit polyclonal antibody against BAT3 protein. The molecular weight of BAT3 was ∼122 kDa. Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blots. B. Total RNA was isolated from cells; cDNA was prepared and subjected to real-time PCR for BAT3 gene amplification. ΔΔCT values were normalized to mouse GAPDH gene. C. The exosomal pellet (Ex) and soluble fractions (SF), of both J774A.1 cells and bone marrow-derived macrophages (BMDM) culture supernatants were collected. Total 25 µg of protein from each sample was run on a 12% SDS-PAGE gel followed by western blotting to detect BAT3 (upper panel) and HSP70 (lower panel). The cytoplasmic marker GAPDH and nuclear marker histone H1 served as positive controls for the cytoplasmic extracts and nuclear extracts, respectively, in western blotting experiments.