Detection of thymopoietin-responsive proteins in nude mouse spleen cells by two-dimensional polyacrylamide gel electrophoresis and image processing

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Abstract
Lymphoid cells isolated from the spleen of BALB/c nu/nu nude mice were treated with synthetic human thymopoietin, and newly synthesized proteins were labeled by [35S]methionine incorporation. In the control experiment, the same lot of spleen cells were incubated in the labeling medium without the addition of thymopoietin. Urea/detergent‐soluble proteins were extracted from the cells after 3 h incubation to be separated by two‐dimensional polyacrylamide gel electrophoresis. Spots of [35S]methionine‐labeled proteins were visualized by autoradiography and analyzed by image processing. The computer‐aided spot matching screened out three major thymopoietin‐responsive proteins, TRP‐1, −2 and −3. [35S]Methionine incorporation into TRP‐3, of which the isoelectric point and molecular mass were approximately pI5 and 10 kDa, respectively, was decreased by the thymopoietin treatment. In contrast with the down regulation, TRP‐1, which was slightly higher in pI and slightly larger in molecular mass, and TRP‐2, which was slightly higher in pI and almost the same in molecular mass as TRP‐3, were evidently induced by the treatment. However, TRPs could not be assigned to Thy‐1 antigen on the difference in molecular mass. The specific induction by the thymopoietin treatment suggested that TRP‐1 and −2 might be novel proteins related to the intracellular signal transduction.