Figure 3. PKC⍺ and Cyclin B1 accumulate into the nuclei and interact at G2/M.

(a) Cells were synchronized in G0/G1 by HBSS (G0/G1), G1/S by complete RPMI 10% FBS (G1/S) and G2/M by Nocodazole (G2/M). (b) Immunoblot analysis of total, nuclear and cytoplasmic lysates of cells synchronized at different cell cycle phases was performed to investigat PKC⍺ and Cyclin B1 expression and localization during cell cycle. PKC⍺ and Cyclin B1 increase and translocate into the nuclei during cell cycle, peaking at G2/M checkpoint. (c) Cells were synchronized as a) and analyzed using immunocytochemistry to study PKC⍺ and Cyclin B1 localization. The two enzymes co-localize during cell cycle progression and accumulate into the nuclei in particular at G2/M transition. (d) Co-immunoprecipitations experiments in cells synchronized at G2/M (G2/M) indicated the possibility for PKC⍺ and Cyclin B1 to complex. As negative control non-specific IgG were used (IgG). 50μg total lysate of K562 were used as positive control (Control +). To show that the experiments were performed correctly, immunoblots of the immunoprecipitated proteins are provided. All the experiments were performed more than four times. (e) Cells were blocked at G2/M checkpoint and PKC⍺ or Cyclin B1 were immunoprecipitated. An antibody anti-cdk1/cdc2 was used to detect the capacity for both the proteins to interact with this kinase. (f) The interaction between Cyclin B1 and PKC⍺ takes place in the cytoplasms. Nuclei and cytoplasms of cells synchronized in G2/M were separated. Cyclin B1 was immunoprecipitated and PKC⍺ was detected via Western Blotting. The complex between the two enzymes was appreciable only in the cytoplasmic fractions of the cells. The experiment was repeated three times as shown in the figure.