Fig. 4. Phosphorylation of stathmin at Ser16 is regulated by neural activity in PCs. Immunocytochemistry of primary cultured PCs at DIV-9 was carried out with anti-calbindin and anti-stathmin-phospho-Ser16 antibodies. (A) KN-93 (5 µM) or TTX (1 µM) was supplied from DIV-7 to -9. (C,E) PC cultures were treated with DMSO, 10 µM nifedipine (C), or 30 µM CPCCOEt (E) from DIV-7 to -9. Bars, 20 µm. (B) Quantification of stathmin-phospho-Ser16 signal intensity. The average signal intensity of phospho-Ser16 was normalized to that of calbindin. (D,F) Signal intensity of stathmin-phospho-Ser16 was quantified as in B. Data were obtained from three sister cultures, and P values derived from Student's t-test are shown in the graphs. n, number of PCs measured. In addition to PCs, our culture contained granule cells and glial cells. We therefore employed immunocytochemistry, rather than western blot, to measure the level of phospho-Ser16 in PCs. (G) Immunocytochemical analyses of primarily cultured PCs at DIV-9 were performed with anti-calbindin and anti-stathmin antibodies. Treatment with each reagent was performed as in A, C and E. There are no obvious differences in the stathmin signal level in PCs. Bars, 20 µm. (H) Signal intensity of stathmin was quantified as in B. n, number of PCs measured.