Human Calmodulin Methyltransferase: Expression, Activity on Calmodulin, and Hsp90 Dependence
Figure 4
(A) Lysates of HEK293 cells transiently transfected with FLAG- CaM KMT or FLAG were immunoprecipitated with anti-FLAG antibody. The precipitated proteins were subjected to SDS-PAGE and then Coomassie stained. Molecular mass markers in kDa are indicated on the left. The band of approximately 90 kDa (shown with the asterisk) was excised from the gel, and analyzed by mass spectrometry. The heavy chains of the antibodies ∼50 kDa, two nonspecific bound proteins about 70 kDa and FLAG-CaM KMT immunoprecipitated protein were also observed. (B) Alignment of the protein sequences Hsp90α and HSP90β. The bold stretches of amino acids (26% of the protein sequence) represent peptide sequences as identified by mass spectrometry in the NCBI data bank matching Hsp90α and Hsp90β. Diverse amino acids in Hsp90α and Hsp90β, present in the sequenced peptides and enable to distinguish between the isoforms (shown in red). (C) CaM KMT and Hsp90 proteins immunoprecipitate each other.HEK293 cells were transiently transfected with Myc-CaM KMT or an empty Myc vector and 48 h after the transfection, equal protein amounts of whole cell lysates were immunoprecipitated using an anti-Myc (left), anti-Hsp90 (right) and mock IgG antibody (left) as a negative control. The immunoprecipitates were subjected to the Western blot analysis using anti-Myc and anti-Hsp90 antibody as indicated. Equal protein amounts in the immunoprecipitation assays were demonstrated by analysis of 1% input. These experiments were repeated three times with identical results.