Fig. 4. Phosphorylation state of the Na⁺, K⁺-pump during apoptotic process. The protein phosphorylation level of the Na⁺, K⁺-pump was assessed using the anti-phosphorylated protein antibody. (A) In the left column, cell lysates were directly subjected to SDS-PAGE; anti-Na⁺, K⁺-ATPase 3 subunit antibody (anti-3) was used as primary antibody. It demonstrated the specific binding of the anti-3 antibody to the protein. In the last two columns, an anti-3 or anti-phosphorylated protein antibody (anti-pan) was used as antibody in immunoprecipitation. Western blotting showed a clear 3 subunit protein band. (B) Cortical neurons were treated for 9 hours in control medium, serum-free medium (SD) or in 0.1 µM staurosporine (Staur). Anti-pan antibody was used to precipitate phosphorylated 3 subunit. Antibodies were linked to saturated amount of protein A-sepharose beads. Using anti-3 as primary antibody, western-blotting showed reduced phosphorylation level of the 3 subunit after serum deprivation or exposure to staurosporine. (C) Protein phosphorylation levels of the 3 subunit was measured by band relative gray intensity and corrected by corresponding protein A band intensity. Both serum deprivation (9 hours) and staurosporine exposure (9 hours) reduced the phosphorylation. As a control, more matured neuronal cultures of more than 15 days in vitro were subjected to serum deprivation (9 hours) and showed no decreased phosphorylation state (data not shown), consistent with diminished apoptosis in these cells. n=8 independent experiments for serum deprivation and n=3 for staurosporine. *P<0.05 compared with controls.