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. 2013 Oct 9;110(45):18232–18237. doi: 10.1073/pnas.1308253110

Fig. 2.

Fig. 2.

Agonist-induced ALX homodimerization. (A) Homodimerization of ALX as demonstrated using co-IP. HEK293 cells were transfected with either ALX × 3Flag, ALX × 3HA, or both ALX × 3Flag and ALX × 3HA constructs. Twenty-four hours later, cells were stimulated with AnxA1 (10−8 M), SAA (10−7 M), or Ac2-26 (10−5 M) for 10 min. Immunoprecipitation of ALX × 3HA using anti-HA agarose beads coprecipitated ALX × 3Flag as determined by immunoblotting with anti-Flag antibody. Blots are representative of three experiments. (B) Homodimerization of ALX using BRET. HEK293 cells, transfected with ALX-Rluc and ALX-EYFP, were stimulated with the indicated agonists as in A. Data (mean ± SEM of three experiments) calculated against basal ALX/ALX values. *P < 0.05, ***P < 0.001 vs. vehicle-treated cells (white bar). (C) Examples of real-time BRET analysis between ALX-Rluc and ALX-EYFP expressing HEK293 cells upon application of vehicle (Ctrl), AnxA1 (10−8 M), Ac2-26 (10−5 M), or SAA (10−7 M), as monitored for up to 12 min.