FIG. 3.

Mrj interacts with class II HDACs. (A) Deacetylase activity assay from immunoprecipitation reactions against Mrj (c-Myc antibody) or a nonspecific antibody (HA) in cardiomyocytes infected with AdMrj or a control adenovirus (AdβGal). (B) Transfection assay showing NFAT-dependent activity (9xNFAT luciferase reporter) in the presence of cotransfected NFATc3 and HDAC4/HDAC5/HDAC7/HDAC9 or Mrj. Western blotting is shown below for each transfection reaction to verify expression of HDAC4/HDAC5/HDAC7/HDAC9 (all are flag tagged) or NFATc3. (C) Coimmunoprecipitation assay followed by Western blotting for Mrj by using a c-Myc antibody (upper panel). Reactions were performed using the inputs shown (lower panels) from cells infected with AdHDAC4 alone, AdMrj alone, or both. The polyhistidine antibody served as a positive control for Mrj precipitation, the HA antibody was a negative control, and the Flag antibody showed a specific interaction between Mrj and HDAC4. (D) Coimmunoprecipitation assay followed by Western blotting for HDAC4 and NFATc3 from cotransfected Cos-7 cells with HDAC4, NFATc3, ΔCnA, and Mrj. Mrj was immunoprecipitated with a c-Myc antibody, which identified HDAC4 and NFATc3. (E) SDS-PAGE of [³⁵S]methionine-labeled HDAC4 precipitated with GST, GST-Mrj, or GST-NFATc3 with amino acids 700 to 1075 (GST-NFΔ700-1075). Thirty percent of the input is shown. (F) Coimmunoprecipitation assay with Mrj antibody (c-Myc tag) or a nonspecific IgG, followed by Western blotting for HDAC5 or HDAC7. Cos-7 cells were first cotransfected with HDAC5 or HDAC7 in combination with Mrj. (G) Immunoprecipitation of endogenous HDAC4 (HD4), NFATc3 (NFc3), or nonspecific IgG from C2C12 protein lysates, followed by Western blotting for endogenous Mrj. (H) SDS-PAGE of [³⁵S]methionine-labeled HDAC4 (amino acids 628 to 1084) in the presence or absence of cotranslated Mrj. These translated products were subjected to pull-down assay with GST or GST-NFATc3 (amino acids 405 to 700).