Identification of PDZ Domain Containing Proteins Interacting with 1.2 and PMCA4b
Figure 3
Coimmunoprecipitation of Cav1.2. (a) Coimmunoprecipitation demonstrating an interaction of Cav1.2 with CASK; CASK was expressed in HEK 293 cells, which we additionally transfected with pcDNA3-Cav1.2. We also probed mouse brain lysates. These lysates were precipitated with polyclonal α-Cav1.2 antibody and probed with monoclonal α-CASK antibody during immunoblotting (IB). The positive control (input) consisted of 20 μg of the HEK 293 lysate. The negative control was HEK 293 cells immunoprecipitated (IP) with an irrelevant antibody (α-NFATc2). (b) Interaction between Cav1.2 and NHERF1. The positive control was HEK 293 cells transfected with pcDNA3-NHERF1, and the negative control was incubated with an irrelevant antibody (α-AT2). We precipitated HEK 293 cells stably expressing NHERF1 with polyclonal α-Cav1.2 antibody, and also tissue lysates of heart and kidney. For IB we used α-NHERF1 antibody. (c) IP demonstrated an interaction of Cav1.2α with MAGI-3. Positive and negative controls are as described above. Cav1.2 antibody was used for IP and MAGI-3 antibody for IB. (d) Interaction between Cav1.2 and ZO-1. ZO-1 protein is expressed in ECV cells, hence the positive control was nontransfected ECV cells; the negative control contained ECV cells immunoprecipitated with an irrelevant antibody (α-NFATc2), and for the IP we used Cav1.2 antibody for precipitation and α-ZO-1 for the IB. (e) HEK 293 cells with stable overexpression of α and β subunits of Cav1.2 transfected with HA-KD (36 kDa) or HA-KD+PDZ domain of MAST-205 (77 kDa) were incubated with α-Cav1.2 and protein complexes were subsequently precipitated with protein A/G beads. Western blots were probed with HA antibodies. Irrelevant antibodies were used in negative controls and nonprecipitated organ lysates as positive controls.
