Expression of Erk5 in myeloma cells. (A) Schematic representation of Erk5, with the epitopes recognized by the anti-Erk5 (C-terminal), anti-pErk5, and anti-HA antibodies. (B) Expression of Erk5 in MM cell lines. Erk5 was immunoprecipitated from 1-mg cell extracts and detected by Western blotting with the anti–C-terminus antibody. The position of the Mr markers is indicated at the right. In parallel, and as a loading control, 20 μg protein from each of the cell extracts was analyzed for tubulin content by Western blotting. (C) Specificity of the anti-Erk5 antibody; 1 mg MM1S cell extracts was immunoprecipitated with the anti-Erk5 C-terminus antibody either in the absence or presence of 10 μg of the peptide used to raise the antibody. Erk5 was detected by Western blotting as described in panel B. (D) Expression of Erk5 in patient myeloma cells. Cells were isolated from myeloma patients, plated in complete media, and analyzed by Western blotting for Erk5. The proportion of malignant plasma cells was greater than 90% as indicated by phenotypic analysis before the experiment. (E) Immunofluorescent localization of Erk5 in myeloma cells. Cells were plated on poly-l-lysine–coated coverslips and immunostained with the anti-Erk5 C-terminal antibody that was previously preincubated with the antigenic peptide, where indicated. Erk5 is shown in red, together with a nuclear stain (SYBR green). Bar represents 10 μm.
