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. 2007 Mar 8;26(7):1761–1771. doi: 10.1038/sj.emboj.7601625

Figure 4.

Figure 4

Gephyrin interacts with Pin1 in vitro. (A) Immunofluorescence assay to determine the subcellular distribution of gephyrin WT, Mut-C (see above) and Pin1-FLAG ectopically expressed in HEK 293 cells. In single transfection experiments, gephyrin-GFP and Mut-C-GFP were revealed by the intrinsic green fluorescence of GFP. Pin1-FLAG was visualised by anti-FLAG antibody, followed by TRITC-conjugated secondary antibody. Scale bar, 10 μm. (B) Cotransfection experiments with gephyrin-GFP and Pin1-FLAG. (C) Cotransfection experiments with gephyrin Mut-C-GFP and Pin1-FLAG. (D) Lysates of HEK 293 cells transfected with Pin1WT in the presence of gephyrin FLAG or with the vector alone (as a negative control) were immunoprecipitated with monoclonal antibodies anti-FLAG (lanes 4 and 5). Immunoprecipitates were analysed by Western blotting using anti-gephyrin and anti-Pin1 antibodies. Efficient dephosphorylation of gephyrin upon CIP treatment was verified by Western blot on total lysates and on immunoprecipitated gephyrin-FLAG with MPM-2 antibody (7–8 and 10–11, respectively.) (E) Co-immunoprecipitation experiment on mouse brain lysates using a polyclonal anti-gephyrin antibody and NRS as negative control (lanes 2 and 3, respectively). Immunoprecipitates were analysed by Western blotting using a monoclonal antibody anti-gephyrin and a polyclonal antibody against Pin1.