Figure 2.

SOD1 is phosphorylated in nodularin-treated apoptotic hepatocytes. Primary hepatocytes in suspension culture were pre-labeled for 35 min with ³²Pi and exposed to nodularin (200 nM) for 10 min. Hepatocyte extract was fractionated and the cytosolic fraction (2.5 mg protein) was separated by two-dimensional gel electrophoresis (pI 5.3–6.5, 13 cm). Spots were identified using MALDI MS or MS/MS. See Experimental Section for further details. The figure shows Coomassie-stained gels (A and B) and their corresponding autoradiographs (C and D). Only non-phosphorylated SOD1 (spot N1) was detectable with Coomassie staining. Spots were therefore cut from the Coomassie-stained gels using the autoradiographs as guidance. In nodularin-treated hepatocytes SOD1 was phosphorylated and appeared as two novel spots with an acidic shift (spot P1 and P2). N1, P1 and P2 were identified as SOD1.