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. 2012 Apr 27;7(4):e35735. doi: 10.1371/journal.pone.0035735

Figure 1. Nedd4.2 interacts with AMOTL1 and mislocalizes AMOTL1 from tight junctions.

Figure 1

A, Nedd4.2 reduces AMOTL1 protein levels. Co-expression of myc-tagged Nedd4.2 (myc.Nedd4.2) decreased Flag-tagged AMOTL1 (F.AMOTL1), occasionally to very low protein levels (lane 2). B, AMOTL1 interacts with Nedd4.2. HEK 293T cells were transfected with constructs encoding F.AMOTL1, myc.GFP, or myc.Nedd4.2. Cell lysates were probed with anti-myc and anti-Flag antibodies. Precipitation of F.AMOTL1 immobilized myc.Nedd4.2, but not myc.GFP, while precipitated F.GFP did not bind myc.Nedd4.2. C, Binding of endogenous AMOTL1 to Nedd4.2 was detected with anti-AMOTL1 and anti-Nedd4.2 antibodies, respectively. CEP164 was used as a negative control. D, AMOTL1 co-localizes mostly at the plasma membrane with Nedd4.2 in HEK293T cells. E, MDCK cells were transfected either with empty vector or Nedd4.2. Cells were then stained for endogenous AMOTL1 (grayscale) and the tight junction marker, ZO-1 (red). AMOTL1 is localized to tight junctions together, with ZO-1. Note that in dividing cells (yellow arrowheads) less ZO-1 as well as AMOTL1 are observed at junctions. When Nedd4.2 is added, endogenous ZO-1 as well as AMOTL1 are mislocalized from tight junctions and appear mostly in the cytoplasm. Scale bars represent 20 µm.