Fig. 1. Recombinant hDNMT expression and binding analysis in vivo. (A) Construct used for the expression of DNMTs in Sf9 cells. hDNMT1 expression was driven by the baculovirus p10 promoter. In the same construct, the polyhedrin (pPH) promoter expressed hDNMT3a or hDNMT3b, as indicated by the arrow. (B) Specificity of hDNMT3 antibodies. Proteins, hDNMT3a or hDNMT3b are indicated at the top, and the antibody used at the bottom, of each panel. Each lane had 0.4 µg of purified antigen. Molecular weight markers are shown on the left. (C) Western blot analysis of the expression of the hDNMTs in insect cell extracts. Extracts from Sf9 cells expressing hDNMT3a alone, hDNMT3b alone, or DNMT1 plus hDNMT3a or hDNMT3b, are indicated at the top, and the antibody used at the bottom, of each panel. The Sf9 lane is a control cell extract. (D) Co-immunoprecipitation with anti-DNMTs in Sf9 cells expressing recombinant enzyme(s). Antibodies used for immunoprecipitation are indicated at the top. Monoclonal anti-GFP was used as a control. Insect cell extracts expressing hDNMT1 and hDNMT3a were immunoprecipitated with anti-hDNMT3a, and extracts from cells expressing hDNMT1 and DNMT3b were immunoprecipitated with anti-hDNMT3b. Pre-immune serum is marked as PI. The arrow shows the relative position of hDNMT1, along with purified hDNMT1 as a positive control. (E) Maintenance methyltransferase activity of co-immunoprecipitates with de novo enzymes. Extracts from Sf9 cells expressing hDNMT1 and hDNMT3a (left), or hDNMT1 and hDNMT3b (right), were immunoprecipitated with anti-DNMTs antibodies, as indicated. The immunoprecipitated product was assayed for hDNMT1 activity using poly (dI–dC)·poly (dI–dC) substrate DNA and tritiated AdoMet.