Figure 4

Analysis of total GSK3 expression prior to and following serum-shock. To verify that effects observed at the level of transcription in clones A1.4 and A1.6 corresponded to an expected level of GSK3 knockdown, at the indicated time points, protein and RNA samples were simultaneously isolated from harvested wild-type, A1.4, and A1.6 cells in order to monitor levels of GSK3 expression prior to and following serum shock. RNA samples were subsequently used for reverse-transcription PCR analysis (as seen in Fig. 3), while the protein samples were subjected to Western blot analysis. Protein samples were harvested at TP0, TP4, TP24, TP32 and TP48, and blotted for total GSK3. β-Actin levels were used as a loading control.