Figure 6. Merlin links the AJ and Par3 complexes in vivo and in vitro.

(A,B) Par3 (180kd isoform) was immunoprecipitated from (A) scr-shRNA- or (B) Nf2-shRNA-expressing PAM212 keratinocytes that had been stimulated with calcium for the indicated times. Immunoblotting revealed that Par3 associates with AJ and TJ proteins in the presence but not absence of endogenous Merlin. NRS, normal rabbit serum.

(C) FL-, NT- and CT-Merlin-HA produced by IVTT were mixed with Myc-Par3 produced in Sf9 cells. Immunoprecipitation using an anti-HA antibody and immunoblotting revealed that FL-, NT- and CT-Merlin-HA all associated with Par3 in vitro. 10% of the input mixture is shown in the right panel.

(D) Myc-Par3 produced in Sf9 cells was mixed with IVTT-produced FL-, NT- or CT-Merlin-HA and GST-α-catenin. Complexes were isolated using GST beads; immunoblotting with anti-GST, -Myc, or -HA antibodies revealed that FL- or NT-Merlin-HA, which bind to GST-α-catenin in vitro, but not CT-Merlin-HA, is sufficient to promote Myc-Par3:α-catenin association in vitro.

(E) Par3 was immunoprecipitated from Nf2-shRNA-expressing PAM212 keratinocytes that had been infected with a Merlin-NT-expressing adenovirus and stimulated with calcium for the indicated times. Immunoblotting revealed that reintroduction of Merlin-NT rescues the association of Par3 with AJ and TJ proteins in keratinocytes. NRS, normal rabbit serum; also see Figure S4.