Figure 5

Effect of treatment on CD74 and CD44 proteins A. MIF pull down experiment – Cell lysates from 24 h control cultures were immunoprecipitated with CD74 antibody (lane 1) or CD44 antibody (lane 2,5) and the protein G binding protein complexes separated by polyacrylamide gel electrophoresis, followed by Western blotting using polyclonal anti-MIF or CD74 antibody. Lane 1, CD74 antibody; lane 2, CD44 antibody; lane 3, non-specific antibody; lane 4, no antibody added; lane 5, CD44 antibody. B. CD 74 Western blot analysis – Proteins in cell lysates from 24 h cultures treated with indicated reagents were separated by polyacrylamide gel electrophoresis, followed by Western blotting using polyclonal anti-CD74 antibody. Bands were quantified by digital imaging and are expressed as integrated density values (band area × relative intensity) and are presented as the mean ± SEM of three separate experiments of quadruplicate cultures. All treatments increased the levels of CD74 in the cell lysates. C. CD 74 Western blot – Proteins in cell lysates from 24 h cultures treated with indicated reagents were separated by polyacrylamide gel electrophoresis, followed by Western blotting using polyclonal anti-CD74 antibody. Blot is representative of those from three separate experiments. D. CD 44 Western blot analysis – Proteins in cell lysates from 24 h cultures treated with indicated reagents were separated by polyacrylamide gel electrophoresis, followed by Western blotting using polyclonal anti-CD44 antibody. Bands were quantified by digital imaging and are expressed as integrated density values (band area × relative intensity) and are presented as the mean ± SEM of three separate experiments of quadruplicate cultures. HA and anti-MIF treatments significantly decreased the levels of CD44 in cell lysates. MIF antisense had no effect. E. CD 44 Western blot – Proteins in cell lysates from 24 h cultures treated with indicated reagents were separated by polyacrylamide gel electrophoresis, followed by Western blotting using polyclonal anti-CD44 antibody. Blot is representative of those from three separate experiments.(** – p < 0.01, *** – p < 0.001).