Fig. 9. Gel electrophoretic analysis of CKs in AK 13-1 cells treated with OV. (A) A picture of an immunoblot of SDS polyacrylamide gel (10%) containing high salt pellet fractions (P) and equal amounts of corresponding supernatant fractions (S) that were obtained either from untreated control cells (C), cells treated with either 5 mM OV for 10 minutes (OV) or 0.1 µg/ml okadaic acid for 105 minutes (OA) and enriched mitotic cells (mit) after reaction with CK antibody K_s 13.1 and secondary HRP-coupled antibodies in combination with an enhanced chemiluminescence system. Note the presence of CK13 (lower band) and HK13-EGFP (upper band) in the soluble fraction of cells treated with okadaic acid and in mitotic cells in contrast to untreated and OV-treated cells. The position and relative molecular mass of co-electrophoresed marker proteins are given in units of 1000 in left margin. (B,C) Detection of Coomassie brilliant blue stained polypeptides contained in high salt pellet fractions after separation by two-dimensional gel electrophoresis obtained from untreated control cells (B) and cells treated with 5 mM OV for 10 minutes (C). Note the similar distribution of CK polypeptides (^*, positions of chimera HK13-EGFP) with some minor differences. (D) Immunoblot reaction of high salt pellet fractions of AK13-1 cells without any treatment or after incubation in 5 mM OV for 10 minutes (OV) and 0.1 µg/ml OA for 4 hours (OA) using phospho-epitope specific antibodies LJ4 detecting CK8 pS73, 5B3 reacting with CK8 pS431, and IB4 detecting CK18 pS33 as well as CK antibody L2A1 detecting all CK8, 18 and 19 polypeptides irrespective of their phosphorylation status. The position and relative molecular mass of marker proteins are shown on the left.

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