Fig. 4

HSP60 is secreted by microglia in the surrounding medium and interacts with TLR4 during inflammation. a N9 murine microglial cells were treated with IL-1β for different time periods (3, 6, and 12 h), and the proteins in the used medium were precipitated with trichloroacetic acid (TCA). Western blotting was performed to determine the levels of HSP60 in secretome. Normalization was performed with Ponceau-stained bands. Right panel shows the bar diagram representing mean fold changes in the level of HSP60 with respect to control N9 cells. Twenty microgram of the secreted protein was loaded for western blot of HSP60. b Co-immunoprecipitation analysis of the interaction between HSP60 and TLR4 in cells treated with IL-1β for 3 h. Whole-cell extracts (500 μg) of untreated and treated N9 microglial cells were immunoprecipitated with anti-HSP60 and anti-IgG antibodies and analyzed by western blot analysis with anti-TLR4 antibody (left panel). Right panel (c) shows the western blots with 100 μg of lysates for the detection of total HSP60, TLR4, and β-actin in the IL-1β-treated cells as compared to control cells. Lower panel (b, c) represents bar diagrams which depict mean fold change in the expression of HSP60 and TLR4 in comparison to control in immunoprecipitate (b) and lysate (c), respectively. Representative blots of the three independent experiments are shown here. *p < 0.05, **p < 0.01 in comparison to control values. Data represented are mean ± SD of three independent experiments