Fig. 1. Subcellular distribution of RII in cell lines expressing wild-type or mutant (T54E) RII. (A) Analysis of RII expression in stably transfected cell lines. Interphase cells (2x10⁶ cells per lane) were subjected to SDS-PAGE and immunoblotting with a polyclonal antibody against human RII. Lanes: 1, wild-type Reh (RII-deficient); 2, Reh-pMEP4 (vector transfected); 3, Reh-RII; 4, Reh-RII(T54E). (B) RII was immunoprecipitated from interphase lysates (4x10⁷ cells per lane) of Reh-RII (lane 2) and Reh-RII(T54E) (lane 3) cells. Precipitation from Reh was performed as control (lane 1). Half of the immune precipitates were immunoblotted using a anti-RII polyclonal antibody (upper panel). The other half was incubated for 45 minutes at 22°C in EBS phosphorylation buffer with [-³²P]ATP in the presence of CDK1 (9 pmol minute^-1 µl^-1), separated by SDS-PAGE, dried and subjected to autoradiography (lower panel). The positions of non-phosphorylated (51 kDa) and phosphorylated (53 kDa) RII are indicated. (C-E) Interphase (I) and mitotic (M) Reh cell lines (C), mouse A9 fibroblasts (D) and primary cultures of peritubular cells prepared from rat testes (fibroblast-like) (E) were analyzed by immunofluorescence using anti-RII (upper panel; red in C and D, green in E) or anti-RIIß mAbs (lower panel; green in E) and an affinity-purified polyclonal antibody against AKAP450 (green in C and D, red in E). DNA was stained with Hoechst 33342 (blue). Arrows indicate mitotic centrosomes. Bar: 10 µm. (F) CDK1 phosphorylation of purified recombinant human (lane 1) and bovine (lane 2) RII (150 ng of each). The positions of phosphorylated RII (lanes 1 and 2) and autophosphorylated cyclin B (49 kDa, lane 3) are indicated.