Western immunoblotting and immunofluorescence microscopy with murine monoclonal anti-codanin-1 (COD53, COD177, and COD187) antibodies. (A) Western (full-range) blot with the 3 uncloned hybridoma supernatants. Lanes correspond to HeLa, MEG-01, MEL, and K562 cell line protein extracts. (B) Western blot of whole-cell extracts of mESCs (E14) probed with the COD177 antibody. Bands of approximately 140 kDa (wild-type codanin-1) and more than 250 kDa (codanin-1/β-geo fusion) are visible. This demonstrates the specificity of the monoclonal antibody against the wild-type codanin-1 and a fusion protein of the expected size generated by gene-trapping one allele of Cdan1 (“A mouse model with a gene-trap plasmid in the Cdan1 gene”). (C) Western blot of nonerythroid brain cell line GMMC and HeLa cell line extracts. (D) Comparison of whole-cell extracts (WCE), cytoplasmic (cyto), and nuclear extracts (nuc) from cultured intermediate erythroblasts (ErythroB), and MEL cell line. (E) Immunofluorescence staining of control and CDA-1 primary human erythroblasts using the COD-177 antibody. DNA is counterstained with TOPRO-3.