Fig. 4.

Cholesterol depletion enhances binding between LTβR and modified forms of TRAF2 and NEMO. a Western blot analysis of co-immunoprecipitates of anti-LTβR (IP:LTβR) and control antibodies (IP:IgG) from extracts of A549 cells stimulated with LTα1β2 for 0.5 h upon 1 h preincubation in medium containing either MβCD or vehicle. Antibodies against LTβR, TRAF2 were used for blotting. Input represents 10% of the lysates used for IP. h. e. – high exposure. Graph depicts the analysis of TRAF2 abundance (the main and modified forms of the protein) in LTβR co-immunoprecipitates upon stimulation with LTα1β2. The ratio of co-immunoprecipitated TRAF2 to immunoprecipitated LTβR was quantified. Data were normalized to the TRAF2-LTβR ratio in cells not treated with MβCD, which was assigned a value of 1. Data represent the means ± SEM, n = 3. ns - P > 0.05; *P ≤ 0.05 by one sample t-test. b Lysates of A549 cells preincubated for 1 h with MβCD and then stimulated or not for 0.5 h with LTα1β2 in the presence or the absence of MβCD were analyzed by Western blotting with antibodies against NEMO. h. e. – high exposure. Vinculin was used as a loading control. c Lysates of A549 cells preincubated for 4 h with TAK-243 or vehicle, treated or not for following 1 h with MβCD and then stimulated for 0.5 h with LTα1β2 were analyzed by Western blotting with antibodies against NEMO. h. e. – high exposure. Vinculin was used as a loading control. d Western blot analysis of immunoprecipitation performed as in A. Antibodies against LTβR and NEMO were used for blotting. Input represents 5% of lysates used for IP. Graph shows the abundance of modified NEMO in LTβR immunoprecipitates. Asterisk marks an unspecific band recognized by anti-LTβR antibody. Quantification as in A. Data represent the means ± SEM, n = 3. ns - P > 0.05; *P ≤ 0.05 by one sample t-test. e Lysates of A549 cells transfected with two control (Ctrl) or two TRAF2-targeting siRNAs and stimulated with Ago for 0.5 h were analyzed by Western blotting with antibodies against the indicated proteins. h. e. – high exposure. Vinculin was used as a loading control