Figure 1.

PP1A associates with NACA. A, schematic overview of the proteomics approach used to identify NACA-interacting proteins. B, silver-stained SDS-PAGE gel showing expression and purification of NACA protein. C, mass spectrometry (MS/MS) analysis identified numerous NACA-associated proteins, including several components of the PP1A holoenzyme complex. D, validation of interaction of NACA-FLAG with endogenous PP1A complex proteins. HEK293T cells expressing NACA-FLAG or FLAG empty vector were lysed and immunoprecipitated (IP) with anti-FLAG antibody beads and blotted with anti-FLAG antibody or the indicated PP1A-complex antibodies. E, GST-pulldown analysis showing direct interaction between NACA and PPP1CA as well as PPP1R18. GST-NACA fusion protein or GST alone expressed in bacteria (left panel) was incubated with in vitro translated PPP1R9B, PPP1R12A, PPP1R18, and PPP1CA, and then subjected to SDS-PAGE and Western blot analysis (right panel).