FIGURE 3.

Treatment by IL-1β or an inhibitor of CAMKII increases the association of CD44 with ezrin. A–D, HaCaT cells were treated with either IL-1β (10 ng/ml) or the CAMKII inhibitor KN93 (25 μm) for 20 h, fixed, and stained with primary antibodies against ezrin and the intracytoplasmic domain of CD44 or with corresponding non-immune IgGs (D). Close proximity of CD44 and ezrin was detected with PLA using DNA-labeled secondary antibodies as described under “Experimental Procedures.” Scale bar, 20 μm. E, quantification of the PLA dots per cell (means ± S.E. (error bars), five independent experiments; *, p < 0.05, one-way ANOVA with Dunnett's post hoc test). F and G, cells were treated with IL-1β (10 ng/ml) for 20 h, and protein extracts were subjected to immunoprecipitation with anti-ezrin antibody, followed by Western blotting with anti CD44 and ezrin antibodies. CD44 and ezrin band intensities were measured, and the CD44/ezrin ratio of untreated cultures was set as 100. The data represent means ± S.E. from three independent experiments. H, Western blots of total ezrin and phosphorylated ezrin (pERM) in cells treated with IL-1β (10 ng/ml) for 0.5–2 h. I, quantification of the phospho-ERM/ezrin ratio from the Western blots in H; means and ranges of two independent cultures are shown. J, effect of KN93 on the phospho-ERM/ezrin ratio, quantified from the Western blots of three independent experiments (means ± S.E.).