NF-κB signaling in response to TNFα stimulus is attenuated in BV2 cells overexpressing Hsp72 cells.

<p>A. Western blot assays of BV2 cells stably overexpressing Hsp72 at high levels (Hsp72) and low levels (Hsp72-L) compared with recombinant Hsp72 (Re-Hsp72), control cells (BV2), and control cells transfected with LacZ vector (LacZ). B. NF-κB p65/RelA DNA binding activity is significantly reduced in Hsp72 cells at 15, 20, 30, 105 min time points compared to other cell types. Concentrations were measured with ELISA and normalized with respect to the average measured p65 activity in BV2 cells at 20 min (n = 4 at 150 min for LacZ, n = 6 for all other time points and cell types). C. IKKβ kinase activity following treatment with 10 ng/ml TNFα at 0 min is significantly lower in Hsp72 cells at 0, 2.5, 5, 10 and 20 min. Concentrations were measured with ELISA and normalized with respect to average IKK activation in BV2 cells at 5 min (n = 6 at each time point). D. Total IκBα protein is reduced in Hsp72 cells compared to other cell types at 0, 60 and 90 min. Total IκBα levels were measured using ELISA optical density (OD) readings and normalized with respect to the average OD in BV2 cells at 0 min (n = 9 at each time point). E. Ser-32 phosphorylated IκBα is lower in Hsp72 cells than other cell types at 5, 20, 60 and 90 min. Concentrations were measured with ELISA and normalized with respect to the average level in BV2 cells at 5 min (n = 9 at each time point). F. Western blot assays of total and Ser-32 phosphorylated IκBα protein from whole cell extracts.