FIG. 6.

Analysis of interaction of M53 insertion mutants with M50/p35. (A) Localization of M53 insertion mutants. NIH 3T3 cells were transfected with M53 insertion mutants i104, i115, i128, i131, and i138 alone (first column) or together with wt M50 (second to fourth columns). Single transfected cells were stained with a specific rat antiserum against M53/p38. For detection, a fluorescein isothiocyanate-conjugated secondary antibody was used (green). Cotransfected cells were treated as described above and costained with an M50/p35-specific rabbit serum, which was detected by a Texas red-coupled secondary antibody (red). (B) Pull-down analysis of M53 insertion mutants with Strep-tagged M50/p35. 293 cells were cotransfected with M53 insertion mutants and M50ST. Total cell lysates were analyzed by SDS-PAGE and Western blotting using an M53/p38-specific antiserum (upper panel, T). Proteins in complex with M50ST were precipitated with Strep-Tactin-Sepharose, and desthiobiotin-eluted proteins were separated by SDS-PAGE. Signals for M53/p38 insertion mutants were visualized by Western blotting using a specific antiserum against M53/p38 (lower panel, B).