Figure 5. AmotS176 status impacts binding to the junctional proteins PATJ, Pals1 and E-cadherin.
HEK293-shAmot cells were co-transfected with Flag-PATJ and (A) HA-Amot-WT or (B) HA-Amot-p130S176A or (C) HA-Amot-p130S176E. Total lysates (Input) and Flag or HA IPs were subjected to immunoblot analysis with anti-HA and anti-Flag antibodies, as indicated. (D–E) HEK293-shAmot cells were co-transfected with (D) Flag-Pals1 or (E) E-cadherin and relevant Amot alleles as in panels (A–C). Total lysates (input) and Flag IP were subjected to IB analysis with anti-Amot and anti-Flag antibodies as indicated. (F) HEK293-shAmot cells were transfected with expression vectors for HA-Amot-WT, HA-Amot-p130S176A or HA-Amot-p130S176E. Cells were extracted in F-actin stabilizing buffer and F-actin was pulled down using Biotinylated-phalloidin and streptavidin-coupled magnetic beads. Pull downs were then alayzed by western blotting using anti-Amot or Actin antibodies. The blots shown are representative of three biological replicates (n = 3).