DIFFERENCES IN THE DETECTION OF c-JUN/UBIQUITIN IMMUNOREACTIVE PROTEINS BY DIFFERENT c-JUN ANTIBODIES

Abstract

We compared the immunoreactivity of two widely used c-Jun polyclonal antibodies, SC-45 and Ab-1, in normal mouse hepatocytes and in hepatic tumors by means of immunohistochemistry and Western blotting. The epitope of SC-45 contains Thr 91 and Thr 93, both of which are phosphorylated by c-Jun N-terminal kinase. The epitope of Ab-1 contains Ser 252 which is phosphorylated by glycogen synthase kinase-3 (GSK-3) and casein kinase-II (CKII) and results in the inactivation of c-Jun. In dichloroacetate (DCA)-induced tumors, the protein detected by SC-45 was diffusely spread throughout the cytosol, whereas the protein detected by Ab-1 focused around the nuclei. In the liver tissue adjacent to the DCA-induced tumor, SC-45 stained protein in the nuclei, whereas there was no staining in the nuclei with Ab-1. In DCA-treated mice, SC-45 also detected an increased cytosolic expression of protein in cells that surrounded the central vein. This was not detected by Ab-1. Western blot analysis demonstrated that the c-Jun immunoreactive protein that was highly expressed in DCA-induced tumors was 55 kD rather than 39 kD in molecular weight. Interestingly, the apparent concentrations of the proteins detected at 55 kD varied reciprocally in tumors and in surrounding normal tissue. SC-45 detected higher amounts of the 55 kD protein in nontumor tissue, whereas Ab-1 detected higher amounts of the 55 kD protein in tumor tissue. Furthermore, DCA treatment (2 g/L, 8 weeks) significantly reduced the 55 kD protein detected by Ab-1, but the amount reacting with SC-45 was unchanged. An immunoprecipitation experiment suggested that the 55 kD c-Jun immunoreactive protein also reacted with a ubiquitin antibody. Ab-1 was not able to detect c-Jun protein when it was incubated with CKII, suggesting that the Ab-1 detects a form of c-Jun that has been dephosphorylated in the DNA-binding domain. SC-45 has been shown to detect the dephosphorylated form of c-Jun at Thr 91 and Thr 94. The immunological data obtained using these antibodies indicate that they are interacting with different posttranscriptionally modified forms of c-Jun.