Fig. 1.

C9orf140 is a novel Axin1-interacting protein. a Confirmation of the C9orf140–Axin1 interaction by using co-immunoprecipitation. The 293T cells were co-transfected with Flag-C9orf140/Flag-PSMD4 and HA-Axin1 for 24 h. Cell lysates were subjected to immunoprecipitation with an anti-Flag antibody and an anti-HA antibody. Co-precipitating proteins were visualized by western blotting. The unrelated protein PSMD4 was used as a negative control. b The purified GST or GST-C9orf140-fusion protein bound to agarose beads was added to the lysate of 293T cells ectopically expressing Myc-Axin1. After GST affinity purification, protein complexes were washed and detected by western blot analysis with anti-Myc or anti-GST as indicated. GST protein was used as a negative control. c HeLa cell lysates were immunoprecipitated with rabbit anti-Axin1 antibody, rabbit anti-C9orf140 antibody and control rabbit IgG, then immunoblotted with antibodies as indicated. d C9orf140 co-localizes with Axin1. HeLa cells were transfected with Flag-C9orf140 and Myc-Axin1. The cells were then fixed and stained with anti-Flag antibody (green), anti-Myc antibody (red), and DAPI (blue). No pre-extract: cells were first fixed and then permeabilized as described in the Materials and methods; Pre-extract: cells were first permeabilized and then fixed as described in the Materials and Methods. Bar, 20 μm