Fig. 6.

Dynamin-dependent internalization of LTβR is required for the activation of the alternative but not the classical NF-κB pathway. (A) Flow cytometry analysis of cell surface LTβR expression in untreated and LTα1β2-treated HT29 cells. (B) Colocalization of LTβR and EEA1 to early endosomes. HT29 cells were stimulated with an agonistic antibody to LTβR for the indicated time prior to immunostaining. (C) Recruitment of endogenous AP2μ2 subunit to immunoprecipitated LTβR. (D) Inducible (+ Dox [doxycycline]) rat dominant negative (DN) AP2μ2-expressing HeLa cells transfected with control or siRNA AP2μ2 and stimulated with an agonistic anti-LTβR antibody. (E) HeLa cells transfected with siRNA clathrin heavy chain (CHC) and treated as indicated for the assessment of IκBα degradation and p100 processing. (F) The same cells were transfected with siRNA dynamin-2 and treated as in panel E for the analysis of classical and alternative NF-κB pathways. (G) HeLa cells were treated with the Smac mimetic CmpA prior to staining with the Duolink technology and the indicated antibodies. The endogenous NIK/TRAF3 complex appears in red, and nuclei appear in blue (DAPI).