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. 2012 Feb 20;7(2):e31988. doi: 10.1371/journal.pone.0031988

Figure 8. Co-Immunoprecipitation assays show oligomeric complex formation consisting of EGFP-Vangl1 and 3XHA-Vangl2, or EGFP-Vangl2 and 3XHA-Vangl2.

Figure 8

(A) Immunoprecipitation (IP) of EGFP-Vangl1 or EGFP-Vangl2 using an antibody against GFP pulls down 3XHA-tagged Vangl2 when co-expressed in MDCK cells demonstrating the formation of an oligomeric complex. Similarly, EGFP-Vangl1 or EGFP-Vangl2 IPs also pull down mutant 3XHA-Vangl2S464N protein showing that the Looptail mutation does not disrupt oligomerization. (B,C) Western blots of MDCK cell lysates alone were used to confirm expression of HA-tagged (B) and EGFP-tagged constructs (C), and equilibrate the amount of cell lysates used for immunoprecipitation. Phosphorylated 3XHA-Vangl2 runs in a slower migrating band (arrow, B) and phosphorylated EGFP-Vangl2 generates a doublet (arrowhead, C) in denaturing SDS-PAGE gels of immunoprecipitates or cell lysates. Phosphorylation of 3XHA-Vangl2S464N mutant protein is decreased under all conditions.