Edinburgh Research Explorer Induction of the alternative NF-B pathway by lymphotoxin (LT) relies on internalization of LT receptor

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alter- native NF- (cid:1) B pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin (cid:2) receptor (LT (cid:2) R) as a prototype, we showed that activation of the alternative, but not the classical, NF- (cid:1) B pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LT (cid:2) R essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, internalization (cid:1) internalization mesenteric stromal of alternative NF- (cid:1) B on LT (cid:2) R cellular trafficking as a for specific biological functions of NF- (cid:1) immunoprecipitation experiment (1st IP) by incubation with either control beads or Flag-M2 beads. The immunoprecipitates were then used for detecting Flag-NIK and K48-linked polyubiquitinated NIK. Quantification of signals was per- formed with the Image J software. Oligomerization of LT (cid:2) R and GST pulldown. For studying the ability of LT (cid:3) R to form trimers, we performed transient transfections of HEK 293T cells with a combination of HA-, Flag-, and Myc-tagged LT (cid:3) R expression vectors. A first immunoprecipitation was performed using Flag-M2 agarose beads. The immunoprecipitated material was then selectively released overnight by compe- tition with an excess of 3 (cid:8) Flag peptide (Sigma). The supernatants were subjected to a second immunoprecipitation with either an anti-HA or an anti-Myc antibody for 2 h at 4°C. The immunoprecipitated materials were analyzed by immunoblotting with an anti-Myc or an anti-HA antibody, respectively. To an- alyze aggregation of ectopic LT (cid:3) R, HEK 293T cells were collected 40 h posttransfection, washed twice with phosphate-buffered saline (PBS), and then in- cubated for 30 min at room temperature in PBS containing 1 mM dithiobis[succinimidyl propionate] (DSP; Pierce/Thermo Fisher Scientific). After cross-linking, the cells were washed once in PBS and incubated for 15 min in 20 mM Tris-HCl (pH 7.4)–PBS to stop the reaction. The cells were scraped in SDS buffer, and protein extracts were analyzed by immunoblotting for LT (cid:3) R expression. pGEX-4T1-wt LT (cid:3) R or mutant expression vectors were transformed into Escherichia coli BL21. Bacterial cultures were grown to an A 600 of 0.6 and were induced with 0.5 mM isopropyl- (cid:3)

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