Fig. 3. Expression and characterization of Mi proteins. A, characterization of the serum directed against Mi protein. The murine mi cDNA inserted in the pSG5 expression vector was translated in [35S]methionine-containing reticulocyte lysate. Translated proteins were immunoprecipitated with rabbit serum MiC prepared against bacterially expressed Mi COOH-terminal peptide (Lane 1) and with cognate preimmune serum (Lane 2). Lane 3, translated crude Mi protein. B, detection of Mi by immunoprecipitation. Selected LE-mi-transfected CNR cells or LE-transfected CNR cells were labeled with [35S]methionine/cysteine for 30 min. Labeled cells were solubilized in RIPA buffer. Cell lysates were then immunoprecipitated with the serum MiC (Lanes 2 and 4) and with cognate preimmune serum (Lanes 3 and 5). Lane 1, LE-mi-CNR lysate were first immunoprecipitated with the serum MiC, and proteins recovered from the immunocomplexes bound to protein A-Sepharose were immunoprecipitated again with the same serum. C: a, phase contrast of selected LE-mi-CNR. b, subcellular localization of Mi assayed by indirect immunofluorescence on fixed cells with serum MiC. Anti-Mi-immunoreactive proteins were detected with tetramethylrhodamine isomer R-labeled swine antirabbit immunoglobulins secondary reagent. c, phase contrast of selected LE-CNR used as control. d, subcellular localization of Mi assayed by indirect immunofluorescence. Nuclei are labeled in LE-mi-CNR cells but not in control cells.