Fig. 3. Association of MAGI-3 with RPTPß. (A) HEK-293T cells were transfected with MAGI-3, HA-tagged RPTPß or with both MAGI-3 and RPTPß as indicated (-/+). Cell lysates were subjected to immunoprecipitation with an antibody against the HA-tag that recognizes the extracellular domain of RPTPß (IP:HA) or an antibody to MAGI-3 (IP:MAGI-3) as indicated on the right. Washed immunocomplexes were separated on an SDS gel and immunoblotted with an antibody to HA-tag (left panels) or to MAGI-3 (right panels). (B) Pulldown of MAGI-3 by the cytoplasmic domain of RPTPß. Lysates of HEK-293T cells expressing MAGI-3 were mixed with agarose-bound GST or GST-fusion proteins containing the entire cytoplasmic domain of RPTPß (ßD12), the first phosphatase domain (ßD1), the second phosphatase domain (ßD2) or a mutant form lacking the C-terminus (ßD12dCT) as indicated. Bound proteins were immunoblotted with an antibody to MAGI-3. Immunoprecipitation with an antibody to MAGI-3 (MAGI-3) was used as a control. (C) Interaction of the C-terminal tail of RPTPß with MAGI-3. Rat brain membrane lysate was mixed with immobilized peptides corresponding to the C-terminal tails of RPTPß (RPTPßCT), a peptide containing a scrambled sequence (ßCTS), the C-terminal peptide of Caspr2 (CSP2CT) or a similar peptide lacking the last amino acid (CSP2dCT). Bound proteins were separated on SDS gels and blotted with an antibody to MAGI-3. As a positive control, a lysate sample used in the pulldown experiment was immunoprecipitated with an antibody to MAGI-3 (MAGI-3). Molecular weight markers are shown in kDa on the right. (D) Two-hybrid analysis. The ability of the different domains of RPTPß to interact with a MAGI-3 construct, containing its third and a fourth PDZ domains, was examined using the two-hybrid method (-/+). All RPTPß constructs containing the C-terminal tail interact with MAGI-3, unlike those that lacked this region.