<p>(A) Western blot to detect BAT3 in total lysates and supernatant of heat shock treated monocyte-derived iDCs. (B). ELISA plates were coated with recombinant proteins, buffer control (Neg), NKp46-Ig and NKp30-Ig (concentration of 100 ng/ml), followed by incubation with 100 µl of concentrated supernatant obtained from heat shock treated iDCs and detected with anti-BAT3. Data represents absorbance at 492 nm. (C) Laser Scanning Microscopy to visualize HLA- A, B, C and BAT3 on dendritic cells upon staining with specific primary antibodies and labelled secondary antibodies. HLA-A, B, and C (green), BAT3 (red) and merge (right-yellow). Blue represents Hoechst33342 staining of cell nuclei. (D) Quantitative Real time PCR to detect BAT3 mRNA in 293T and iDCs upon exposure to heat shock. The Y-axis determines the fold change; where the untreated samples were normalized to factor 1. (E) Supernatant was collected from iDCs cells either untreated or treated with non-lethal heat shock (Heat shock) and analysed in specific BAT3 ELISA (sandwich method) to determine the amount of BAT3 in the supernatant. Error bars represent the standard deviation of duplicate samples. One representative experiment of three is shown.</p
