Over-expression of CHIP and BAG-1 increases DALIS formation, whereas HspBP1 and BAG-3 attenuate formation.

<p>(A) Co-chaperone levels were determined by immune blotting with specific antibodies following transfection of RAW309 macrophages with equal amounts of empty pcDNA3.1 plasmid (‘−’) or the same vector containing the coding region for the indicated co-chaperone (‘+’). Cell lysates were prepared 24 h after transfection. (B) RAW309 macrophages were transfected with empty plasmid (‘control’) or an equal amount of a co-chaperone encoding plasmid for 24 h followed by LPS addition for 12 h. DALIS formation was analyzed using the filter trap assay. Detergent insoluble ubiquitin conjugates (‘ub-conj.’) were detected on the filter with FK2 antibody and signal intensity was quantified. Value for LPS-stimulated control cells was set to 1. Error bars represent sem from at least three independent experiments. (C) Representative immune fluorescence micrographs of transfected macrophages after over-expression of the indicated co-chaperones for 24 h followed by LPS stimulation for 12 h. Lower panel shows a quantification of obtained data. Number of DALIS observed in control cells were set to 1. Error bars represent sem from at least three independent experiments. Scale bars: 10 µm.