Fig. 1.

Sequestration of the plasma membrane pool of cholesterol by filipin enhances LTβR-triggered activity of the NF-κB pathway and reduces internalization of the ligand-bound receptor. a Lysates of A549 cells preincubated for 0.5 h with filipin or vehicle and stimulated for 0.5 or 1 h with LTβR agonist (Ago) were analyzed by Western blotting with antibodies against the indicated proteins. Vinculin was used as a loading control. Graphs show densitometric analysis for the indicated proteins from Western blotting (protein levels normalized to vinculin). Values are presented as fold change versus controls - unstimulated and untreated cells (black bars). Data represent the means ± SEM, n ≥ 5; ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by one sample t-test (in grey), Student’s t-test (in black). b Immunofluorescence staining of ligand-bound LTβR and EEA1 upon 0.5 h stimulation with LTβR agonist in A549 cells preincubated for 0.5 h with either vehicle (Veh.) or filipin. Insets: magnified views of boxed regions in the main images. Scale bars, 20 μm. c Analysis of integral intensity and number of LTβR- and EEA1-positive vesicles in cells treated as in B. Values are presented as fold change versus control - vehicle-treated cells marked as a black line, set as 1. Data represent the means ± SEM, n = 5. ns - P > 0.05; *P ≤ 0.05; ***P ≤ 0.001 by one sample t-test