siRNA against GSK3 does not influence cyclin D1 phosphorylation: (A) A vector expressing activated GSK3β protein was injected into an NIH3T3 plate within a circular area marked on the back of the coverslip and indicated in the photographs above. (B) A similar injection of GSK3β was preceded 15 hrs earlier with an injection of an siRNA mixture against GSK3 α and GSK3 β. 5 hrs after these GSK3 plasmid injections cells were fixed and the GSK3 stained with a fluorescent antibody stain. The fluorescence photographs of each injection are presented. (C) To determine the influence of reduced GSK3 expression on the phosphorylation of cyclin D1, this siRNA was microinjected 15 hrs prior to a pulse with BrdU, fixation and staining for phospho-Thr-286 cyclin D1, BrdU and DNA. The average phospho-Thr-286 cyclin D1 levels in each cell cycle phase of injected, or neighboring uninjected cells is presented. MG132 was added 2 hrs prior to fixation to preserve phosphorylated cyclin D1 as above.