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. 2003 Aug;23(16):5790–5802. doi: 10.1128/MCB.23.16.5790-5802.2003

FIG. 7.

FIG. 7.

HSP27 associates with phosphorylated I-κBα. (A) Control-transfected and HSP27-transfected U937 cells were treated as indicated (VP16, 100 μM, 4 h; ΤNF-α, 20 ng/ml, 4 h; MG132, 25 μM) before monitoring of phosphorylated I-κBα (P-I-κBα) expression by Western blotting. HSC70 served as a loading control. (B) Total cell lysates were prepared from HSP27-transfected U937 cells either left untreated or treated for 4 h with etoposide (VP16, 100 μM), ΤNF-α (20 ng/ml), and/or MG132 (25 μM) and then immunoprecipitated (IP) with the indicated antibodies. The indicated proteins were detected by Western blotting. (C) Control-transfected and HSP27-transfected U937 cells and control-transfected and HSP27-transfected MEF cells were transiently transfected with an empty vector or a plasmid containing a nonphosphorylatable, nondegradable mutant form of I-κBα (I-κBα S32A/S36A) (transfection efficiency measured with a β-galactosidase-expressing plasmid, 35 to 40%). At 36 h later, cells were treated with 100 μM etoposide (VP16) or 20 ng of TNF-α/ml for 4 h (U937 cells) or 24 h (MEF cells) before measurement of the percentage of apoptotic cells. Error bars indicate standard deviations (n = 4).