Identification of PDZ Domain Containing Proteins Interacting with 1.2 and PMCA4b
Figure 6
Cav1.2 C-terminal end interaction with PDZ domain of nNOS. (a) Lysates from mouse organs (aorta, brain) and lysates from untransfected HEK 293 cells and HEK 293 cells transfected with pcDNA3-Cav1.2 were incubated with Glutathione-Sepharose beads containing equal amounts of GST (pGEX-4T-3) and the nNOS PDZ domain fused to GST (pGEX-4T-1-nNOS-PDZ). For detection we used α-Cav1.2 (190–210 kDa). An interaction is observed between Cav1.2 and the GST fusion protein pGEX-4T-1-nNOS-PDZ, but not with GST. (b) Lysates were the same as described above. Here the HEK 293 cells were transfected with pcDNA3-nNOS. The negative control was GST (pGEX-4T-3). For detection we used α-nNOS (160 kDa). An interaction between nNOS to the fusion protein pGEX-4T-1-LTCC, where the final C-terminal 10 amino acids were fused to GST, was shown. (c) Coimmunoprecipitation demonstrated an interaction of Cav1.2 with nNOS. HEK 293 cells were transfected with pcDNA3-nNOS. 20 μg of the protein lysate was used directly as input for SDS-polyacrylamide gel electrophoresis. The negative controls were HEK 293 cells transfected with nNOS expression constructs immunoprecipitated with an irrelevant α-rabbit antibody (α-AT2), and the last lane contained HEK 293 cells (stably expressing the α and β subunits of Cav1.2) co-transfected with pcDNA3-nNOS, and immunoprecipitated with α-Cav1.2 antibody. For detection, we used the antibody α-nNOS.
